Anti-viral compounds, pharmaceutical compositions, and methods of use thereof

ABSTRACT

Disclosed herein are compounds, pharmaceutical compositions, and related methods for the treatment of viral infection, including RNA viral infection in subjects. The compounds, pharmaceutical compositions, and methods can modulate the innate immune antiviral response in vertebrate cells, including activating the RIG-I pathway.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 15/885,333 filed Jan. 31, 2018, which is acontinuation of U.S. patent application Ser. No. 15/308,058, filed Oct.31, 2016, now U.S. Pat. No. 9,884,876 issued Feb. 6, 2018, which claimsbenefit of National Phase Application No. PCT/US2015/030014 filed May 8,2015, which claims the benefit of U.S. Provisional Patent ApplicationNo. 61/991,418 filed on May 9, 2014, and U.S. Provisional ApplicationNo. 62/177,900, filed on Mar. 25, 2015, each of which is incorporated byreference herein in their entirety.

FIELD OF THE DISCLOSURE

Compounds, pharmaceutical compositions, and methods disclosed herein areuseful for treating viral infection, including RNA viral infection, insubjects.

BACKGROUND OF THE DISCLOSURE

As a group, RNA viruses represent an enormous public health problem inthe U.S. and worldwide. Well-known RNA viruses include influenza virus(including the avian and swine isolates; also referred to herein asflu), Hepatitis C virus (HCV), Dengue virus (DNV), West Nile virus(WNV), SARS-coronavirus (SARS), MERS-coronavirus (MERS), respiratorysyncytial virus (RSV), and human immunodeficiency virus (HIV). Theseviruses are responsible for pandemic outbreaks and threats to publichealth that have occurred throughout history. Flaviviruses,Henipaviruses, Filoviruses, and Arenaviruses are among emerging RNAviruses that pose significant public health and biodefense threats.These viruses collectively place hundreds of millions of people at riskof infection throughout the world. Many of the emerging RNA virusescause viral hemorrhagic fever and can result in significant morbidityand mortality. Dengue virus (DNV) and West Nile virus (WNV) are bothFlaviviruses (positive strand RNA virus) and Arboviruses, transmittedthrough mosquitoes; thus these viruses represent a potent potentialbiological threat through their ability to transmit readily amonginsects or animals and humans, high infectivity, and their potential tobe weaponized in bioterror events.

At least 4 subtypes of Ebola virus (EV) are infectious to humans (Zaire,Sudan, Bundibugyo, and Cote d'Ivoire). EV outbreaks have been describedin Africa with a fatality rate of up to 90%. Feldmann, H., et al. (2011)Lancet 49, 1-14. Cases of EV infection have been reported in othercountries including, very recently, the United States. The natural hostfor EV is not defined, but nonhuman primates (NHP) are susceptible. EVis a negative-strand RNA virus of the Filoviridae and can be spreadeffectively from person-to-person.

Seasonal flu infects 5-20% of the population annually, resulting in200,000 hospitalizations and 36,000 deaths. Influenza can precipitateviral or secondary bacterial pneumonia, and complicated disease in thoseat the extremes of age or with weakened immune systems. Coronavirusesare common throughout the world and typically cause mild to moderaterespiratory illness, although certain coronaviruses cause severerespiratory illness and death. A 2003 multi-country outbreak ofSARS-coronavirus infection resulted in approximately 8,000 infectionsand nearly 800 deaths. Recently there have been reported cases of MiddleEast Respiratory Syndrome caused by MERS-coronavirus.

More than 170 million people worldwide are infected by HCV, and 130million of these are chronic carriers at risk of developing chronicliver diseases (cirrhosis, carcinoma, and liver failure). As such, HCVis responsible for two thirds of all liver transplants in the developedworld. Recent studies show that the death rate from HCV infection isrising due to the increasing age of chronically infected patients.

DNV is the most prevalent flavivirus in humans, is endemic in mosttropical and subtropical countries, and is currently emerging elsewhereincluding the U.S. and across the Pacific Islands. DNV circulates as 4serotypes (DNV1-4) and following a first infection, re-infection canlead to fatal hemorrhagic fever and shock syndrome. Infection isbelieved to provide life-long immunity against reinfection by the sameserotype, but not against other serotypes. Epidemic outbreaks have beenreported in many countries throughout Latin America, South-East Asia,and the Western Pacific Regions. It is estimated that between 50 and 100million cases of Dengue fever occur globally each year. DengueHemorrhagic Fever and Dengue Shock Syndrome represent severe forms ofthe disease. Currently there is no specific antiviral therapy to treatDNV infection and no approved vaccine.

WNV is a related flavivirus that is endemic in regions of Africa andAsia, but is now emerging in the Western hemisphere. WNV isneuroinvasive to cause serious encephalitis disease and is lethal inabout 6% of cases. Neuroinvasive WNV can present as meningitis,encephalitis or less frequently a flaccid paralysis referred to aspoliomyelitis. WNV was largely absent from North America prior to 1999,but reemerged on the continent following an isolated outbreak ofencephalitis in New York. In the subsequent 7 years, WNV infectionspread throughout the 48 contiguous United States, and current estimatessuggest as many as 2-3 million Americans have been infected. Over thepast 20 years, outbreaks have been reported in parts of Europe, NorthAfrica, the Middle East, and North America. Currently there is nospecific antiviral therapy to treat WNV infection and no approvedvaccine.

Among the RNA viruses listed, very few vaccines are currently approvedfor clinical use. One such vaccine exists for influenza virus, whichmust be revised and administered annually. Accordingly, drug therapy isessential to mitigate the significant morbidity and mortality associatedwith these viruses. Unfortunately, the number of antiviral drugs islimited, many are poorly effective, and nearly all are plagued by therapid evolution of viral resistance and a limited spectrum of action.Ribavirin, a guanine nucleoside analog, has been studied in clinicaltrials of diverse RNA virus infections and is likely the most broadlyacting antiviral agent available. Ribavirin is approved to treatHepatitis C virus (HCV) and respiratory syncytial virus (RSV) infection,and Lassa virus related mortality was shown to be reduced withintravenous ribavirin treatment. However, it is weakly effective as asingle agent and has significant hematologic toxicity. Both classes ofacute influenza antivirals, adamantanes and neuraminidase inhibitors,are only effective within the first 48 hours after infection, therebylimiting the window of opportunity for treatment. High resistance toadamantanes already restricts their use, and massive stockpiling ofneuraminidase inhibitors will eventually lead to overuse and theemergence of resistant strains of influenza.

Based on the foregoing, there is an immense and unmet need for effectivetreatments against viral infections. Most drug development effortsagainst viruses target viral proteins. RNA viruses have small genomes,with many encoding less than a dozen proteins, resulting in a verylimited number of viral targets for new drugs. This is a large part ofthe reason that current drugs are narrow in spectrum and subject to theemergence of viral resistance. However, there is benefit to discovery ofnew viral targets for inhibition. Alternatively, direct-acting antiviraltherapy can work to counteract any infection mechanisms such as viralentry into a host cell.

New antiviral therapy can act directly against viruses. In particular,new antiviral therapy can exploit the fact that these viruses aresusceptible to control by innate intracellular immune defenses thatfunction to suppress virus replication and spread. Compounds that act oncellular targets are likely to be more effective, be less susceptible tothe emergence of viral resistance, cause fewer side effects, and beeffective against a range of different viruses. An effectivebroad-spectrum antiviral, whether used on its own or in combination withother therapies, would be an enormous benefit to current clinicalpractice. While interferon is in principal host-mediated and broadspectrum, many viruses have evolved the ability to disrupt interferonsignaling downstream of drug action at the receptor. An importantcriterion is the development of drugs that activate innate immunesignaling below specific virus countermeasures and are a unique additionto conventional antiviral compounds in development or on the market. Asone such innate immune antiviral response, the RIG-I-like receptor (RLR)pathway of innate antiviral immunity can impose a potent blockade to RNAvirus infection through the actions of a variety of antiviral defensegenes.

SUMMARY OF THE DISCLOSURE

The compounds, pharmaceutical compositions, and methods disclosed hereinshift the focus of viral drug development away from the targeting ofviral proteins to the targeting and enhancing of the host's innateantiviral immune response. The present disclosure relates to compounds,pharmaceutical compositions including the compounds, and associatedmethods of use to treat viral infection, including RNA viral infection.In certain embodiments, the compounds modulate the RIG-I pathway.

Embodiments of the present disclosure can provide compounds representedby the formula

wherein L is NR², O, S, C(═O)N, CR²R³CR²R³, CR²R³NR², CR⁴═CR⁴, CR²R³O,CR²R³S, NR²CR²R³, NR²C(═O), NS(O)_(t), OCR²R³, SCR²R³;

V is (CR²R³)_(u), C(═O)CR²R³, CR²R³O, CR²R³OCR²R³, CR⁴═CR⁴, C(═NR²), orC(═O);

Q is NR², O, S(O)_(t), or a bond;

t=0, 1, 2; u=0-3;

wherein a dashed line indicates the presence or absence of a bond;

R¹ is R^(a), OR², or NR²R³;

each R^(a) is independently H, optionally substituted hydrocarbyl,optionally substituted aryl, optionally substituted heteroaryl;

R² and R³ are each independently R^(a), C(═O)R^(a), SO₂R^(a), or R² andR³ form an optionally substituted carbocyclic, heterocarbocyclic, aryl,or heteroaryl ring;

each R⁴ is independently R², OR^(a), C(═O)R^(a), C(═O)NR²R³, NR²R³,NR^(b)(═O)R^(a), SR^(a), SOR^(a), SO₂R^(a), SO₂NHR^(a), SO₂NR²R³,NCOR^(a), halogen, trihalomethyl, CN, S═O, nitro, or two R⁴ groups forman optionally substituted carbocyclic, heterocarbocyclic, aryl, orheteroaryl ring;

W and X are each independently N, NR^(a), NR^(S), O, S, CR²R⁴ or CR⁴;

R⁵ is R^(a), C(═O)R^(a), SO₂R^(a), or is not present;

Y¹ Y², Y³ and Y⁴ are each independently CR⁴ or N; and

NR²R³ may form an optionally substituted heterocylic or heteroaryl ringincluding pyrrolidine, piperidine, morpholine, and piperazine.

In some embodiments, compounds can be represented by the formula

wherein R⁴ is R^(d), SO₂R^(d), C(═O)R^(d), NHC(═O)R^(d), R^(e), OR^(c),or CF₃, wherein R^(c) is H or C₁-C₁₀ hydrocarbyl, R^(d) is unsubstitutedheterocyclic or unsubstituted carbocyclic, and R^(e) is substitutedheteroaryl or substituted phenyl; and

-   n is 1 or 2.

Some embodiments of the present disclosure can include a pharmaceuticalcomposition including any of the compounds as described herein.

In addition, embodiments of the present disclosure can include methodsof treating a viral infection in a subject including administering tothe subject a therapeutically effective dose of a pharmaceuticalcomposition as described herein thereby treating the viral infection inthe subject.

Further, embodiments of the methods of the present disclosure caninclude administering any of the pharmaceutical compositions describedherein as an adjuvant for a prophylactic or therapeutic vaccine.

Embodiments of the present disclosure include methods of modulating theinnate immune response in a eukaryotic cell, including administering tothe cell any of the compounds as described herein. In some embodimentsthe cell is in vivo. In other embodiments the cell is in vitro.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D show in vitro biological activity. In FIG. 1A, thehigh-throughput screen “hit” compound, compound 1 of Table 1, wasvalidated by demonstrating dose-dependent induction of theIFNβ-luciferase (IFNβ-LUC, left), ISG56-luciferase (ISG56-LUC, center),and the ISG54-luciferase (ISG54-LUC, right) reporter genes. RLU=relativeluciferase units. FIG. 1B confirms the specificity of compound 1, 10 μMof which (compound 1) does not induce the non-specific β-actin promoterrelative to vehicle control (DMSO), and in contrast to equivalent doseof a positive control compound (CPD X). FIG. 1C shows HeLa cells treatedwith increasing amounts of compound 1 showed dose-dependent increase ininterferon regulatory factor (IRF)-3 translocation to the nucleus,quantified by nuclear intensity minus cytoplasmic intensity (“normalizednuclear intensity”). FIG. 1D shows HeLa cells treated with increasingamounts of compound 1 showed dose-dependent increase in NFκBtranslocation, quantified by nuclear intensity minus cytoplasmicintensity. “SeV” refers to Sendai virus infection, the positive control.

FIGS. 2A-2C show induction of gene expression by compound 1 and 2 ofTable 1. FIG. 2A shows gene expression levels of IFIT2 (left) and OAS1(right) in HeLa cells over time from 4-24 hours post treatment with 10μM compound 1 (grey; OAS1 only) or 10 μM compound 2 (black; IFIT2 andOAS1 both shown). FIG. 2B shows gene expression levels of IFIT2 inPH5CH8 cells (solid color bars) and HeLa cells (black checked bars)treated with 10 μM compound 1 (CPD 1) or compound 2 (CPD 2). FIG. 2Cshows gene expression levels of IFIT2 (left), OAS1 (center), and M×A(right) in primary HUVEC cells that were treated with 1 μM compound 1(CPD 1) or 1 μM compound 2 (CPD 2).

FIGS. 3A-3B show induction of gene expression by compound 3 and compound7 of Table 1. FIG. 3A shows IFIT2 gene expression was induced by 5 μMcompound 3 or compound 7. FIG. 3B shows compound 3 induced innate immunegene expression in mouse macrophage cells.

FIG. 4 shows induction of the chemokines IL-8, MCP-1, MIP-1α, and MIP-1βby dendritic cells treated with compound 1 of Table 1 (concentrationsshown in μM). LPS is shown as a positive control inducer of chemokineexpression.

FIG. 5 shows results of experiments performed using the protocol ofExample 8, demonstrating the antiviral activity of select compounds ofFIG. 5 that demonstrated antiviral activity against RSV. +++=greaterthan 70% inhibition of infection, ++=greater than 50% inhibition,+=greater than 30% inhibition, −=less than 30% inhibition.

FIG. 6 shows antiviral activity of example compounds against Influenza Avirus Udorn/72. Treatment of HEK293 cells with increasing concentrationsof compound 3, compound 7, compound 9, and compound 10 of Table 1resulted in dose-dependent inhibition of virus infection (shown as %untreated negative control). Calculated IC50 values are shown.

FIG. 7 shows the antiviral activity of compound 5 and compound 20 ofTable 1 against Dengue virus (DNV) type 2. Treatment with increasingamounts of the compounds showed dose-dependent decrease in infection byvirus.

FIG. 8 shows antiviral activity of example compounds against DNV type 2.Treatment of Huh 7 cells with increasing concentrations of compound 8,compound 3, compound 5, compound 6, compound 7, compound 9, and compound10 of Table 1 resulted in dose-dependent inhibition of virus infection(shown as % untreated negative control). Calculated IC50 values areshown.

FIG. 9 shows antiviral activity of example compounds against humancoronavirus OC43. Treatment with increasing concentrations of compound3, compound 5, compound 6, and compound 7 of Table 1 resulted indose-dependent inhibition of virus infection (shown as % untreatednegative control). Calculated IC50 values are shown.

FIG. 10 shows results from exploratory pharmacokinetic (PK) studies.FIG. 10A,administration of compound 3 of Table 1 via oral (PO) orintravenous (IV) route resulted in detectable levels of compound inserum samples obtained up to 250 minutes post treatment. FIG. 10B, at 4hours post treatment of compound 3 and compound 7 of Table 1, there wasdetectable compound in lung tissue.

FIG. 11A-11C show a study performed using the mouse hepatitis virus type1 (MHV-1) coronavirus model. Treatment with compound 3 of Table 1resulted in decreased pathological symptoms including weight loss FIG.11A and increased survival 11B after lethal challenge with MHV-1. FIG.11C virus was decreased in the lung of animals treated with compound 3.

FIG. 12 shows the in vitro activity of compound 12 of Table 1 of thedisclosure against EBOV at 5 μM, showing greater than a 2 log reductionin EBOV titer in vitro.

FIG. 13 shows the dose response activity of compound 8 of Table 1against DENV-2, as FFU/ml.

DETAILED DESCRIPTION

The present disclosure provides compounds, pharmaceutical compositions,and methods that shift the focus of viral treatments away from thetargeting of viral proteins to targeting and enhancing the host(subject's) innate antiviral response. Such compounds, pharmaceuticalcompositions, and methods are likely to be more effective, lesssusceptible to the emergence of viral resistance, cause fewer sideeffects, and be effective against a range of different viruses.

The retinoic acid-inducible gene 1 (RIG-I) pathway is intimatelyinvolved in regulating the innate immune response to virus infectionsincluding RNA virus infections. RIG-I is a cytosolic pathogenrecognition receptor that is essential for triggering immunity to a widerange of RNA viruses. RIG-I is a double-stranded RNA helicase that bindsto motifs within the RNA virus genome characterized by homopolymericstretches of uridine or polymeric U/A motifs. Binding to RNA induces aconformation change that relieves RIG-I signaling repression by anautologous repressor domain, thus allowing RIG-I to signal downstreamthrough its tandem caspase activation and recruitment domains (CARDs).RIG-I signaling is dependent upon its NTPase activity, but does notrequire the helicase domain. RIG-I signaling is silent in resting cells,and the repressor domain serves as the on-off switch that governssignaling in response to virus infection.

Without being bound by a theory or particular mechanism of action, RIG-Isignaling is transduced through IPS-1 (also known as Cardif, MAVs, andVISA), an essential adaptor protein that resides in the outermitochondrial membrane. IPS-1 recruits a macromolecular signalingcomplex that stimulates the downstream activation of IRF-3, atranscription factor that induces the expression of type I interferons(IFNs) and virus-responsive genes that control infection. Compounds thattrigger RIG-I signaling directly or through modulation of RIG-I pathwaycomponents, including IRF-3, present attractive therapeutic applicationsas antivirals or immune modulators.

In certain embodiments, a high-throughput screening approach was used toidentify compounds that modulate the RIG-I pathway. In particularembodiments, validated RIG-I agonist lead compounds were demonstrated tospecifically activate IRF-3. In additional embodiments, the compoundshave one or more of the following advantages: induce expression ofinterferon-stimulated genes (ISGs low cytotoxicity in cell-based assays,suitable for analog development and SAR studies, drug-likephysiochemical properties, and/or antiviral activity against virusesincluding Dengue virus (DNV), human coronavirus (SARS and MERS-likepathogen), influenza A virus, respiratory syncytial virus (RSV), and/orHepatitis C virus (HCV). In additional embodiments, the compoundsexhibit antiviral activity against dsDNA viruses including humancytomegalovirus. In certain embodiments, the compounds exhibit all ofthese characteristics.

The disclosed compounds represent a new class of antiviral therapeutics.Although the disclosure is not bound by a specific mechanism of actionof the compounds in vivo, the compounds are selected for theirmodulation of innate immune antiviral responses. In certain embodiments,the modulation is activation of the RIG-I pathway. Compounds,pharmaceutical compositions, and methods disclosed herein function todecrease one or more of: viral protein, viral RNA, and infectious virusin laboratory models of viral infection.

The disclosure herein relates to a class of compounds represented byFormula 1:

wherein L can be NR², O, S, C(═O)N, CR²R³CR²R³, CR²R³NR², CR⁴═CR⁴,CR²R³O, CR²R³S, NR²CR²R³, NR²C(═O), NS(O)_(t), OCR²R³, SCR²R³;

-   V is (CR²R³)_(u), C(═O)CR²R³, CR²R³O, CR²R³OCR²R³, CR⁴═CR⁴, C(═NR²),    or C(═O);-   Q can be NR², O, S(O)_(t), or a bond;-   t=0, 1, 2; u=0-3;    wherein a dashed line indicates the presence or absence of a bond;    R¹ can be R^(a), OR² or NR²R³; each R^(a) can independently be H,    optionally substituted hydrocarbyl, optionally substituted aryl,    optionally substituted heteroaryl; R² and R³ can each independently    be R^(a), C(═O)R^(a), or SO₂R^(a), R² and R³ can form an optionally    substituted carbocyclic, heterocarbocyclic, aryl or heteroaryl ring;    each R⁴ can independently be R², OR^(a), C(═O)R^(a), C(═O)NR²R³,    NR²R³, NR^(b)(═O)R^(a), SR^(a), SOR^(a), SO₂R^(a), SO₂NHR^(a),    SO₂NR²R³, NCOR^(a), halogen, trihalomethyl, CN, S=O, or nitro; W and    X can each independently be N, NR^(a), NR^(S), O, S, CR²R⁴ or CR⁴;    R⁵ can be R^(a), C(═O)R^(a), SO₂R^(a), or is not present; Y¹, Y², Y³    and Y⁴can each independently be CR⁴ or N; and NR²R³ can form an    optionally substituted heterocylic or heteroaryl ring including, but    not limited to, pyrrolidine, piperidine, morpholine, and piperazine.

In an embodiment, with respect to Formula 1, Y¹ can be CR⁴ or N. Forexample, Y¹ can be CR⁴.

In an embodiment, with respect to Formula 1, Y² can be CR⁴ or N. Forexample, Y² can be CR⁴.

In an embodiment, with respect to Formula 1, Y² can be CR⁴ or N. Forexample, Y³ can be CR⁴.

In an embodiment, with respect to Formula 1, Y¹ and Y² can both be CR⁴,and in some instances, Y¹ and Y² can form a fused heterocyclic ringoptionally substituted by R⁶ as shown below:

wherein R⁶ can be H, CH₃, CH₂CH₃, Cl, Br, OH, OCH₃, SCH₃, NH₂, NHCH₃,N(CH₃)₂, SO₂NH₂, morpholino, CH₂C≡CH, or NO₂. For example, R⁶ can be Hor CH₃.

In an embodiment, with respect to Formula 1, Y³ and Y⁴ can be CR⁴ or N.For example, Y³ and Y⁴ can be CR⁴.

In an embodiment, with respect to Formula 1, Y¹, Y², Y³ and Y⁴ can beCR⁴. In some instances, Y¹, _(Y2,) Y³ and Y⁴ can be CH. In someinstances, Y¹ and Y² can form a fused heterocyclic ring optionallysubstituted by R⁶, as shown above, and Y³ and Y⁴ can be CH.

The disclosure also relates to a class of compounds represented byFormula 1A.

wherein W can be 0 or S; and R¹ can be R^(a), OR², or NR²R³. R¹, R², R³,R⁴, R^(a), and V and W, can be as defined above with respect toFormula 1. In an embodiment, each R^(a) can independently be H,optionally substituted hydrocarbyl, optionally substituted aryl,optionally substituted heteroaryl; R² and R³ can each independently beR^(a), COR^(a), or SO₂R^(a); each R⁴ can independently be R², OR^(a),NR²R³, SR^(a), SOR^(a), SO₂R^(a), SO₂NHR^(a), NCOR^(a), C(═O)R^(a),CONR²R³, halogen, trihalomethyl, CN, S═O, or nitro; V can be CR²R³,C(═O), C(═O)CR²R³, or C(═N)R²; and W can be O or S. In some instances, Vcan be C═O, and R¹ can be an optionally substituted aryl, or anoptionally substituted heteroaryl. For example, R¹ can be optionallysubstituted phenyl, or R¹ can be optionally substituted naphthyl.

The disclosure also relates to a class of compounds represented byFormula 1B.

wherein W can be O or S; and R¹ can be R^(a) or NR²R³. R¹, R², R³, R⁴,R⁶, R^(a), and V are as defined above with respect to Formula 1.

In some instances, V can be C═O, and R¹ can be an optionally substitutedaryl, or an optionally substituted heteroaryl. For example, R¹ can beoptionally substituted phenyl, or R¹ can be optionally substitutednaphthyl.

The disclosure also relates to a class of compounds represented byFormula 1C.

wherein W can be O or S; and R¹ can be R^(a), OR², or NR²R³. R¹, R², R³,R⁴, R^(a), and V and W, are as defined above with respect to Formula 1.In an embodiment, each R^(a) can independently be H, optionallysubstituted hydrocarbyl, optionally substituted aryl, optionallysubstituted heteroaryl; R² and R³ can each independently be R^(a),C(═O)R^(a), or SO₂R^(a); each R⁴ can independently be R², OR^(a), NR²R³,SR^(a), SOR^(a), SO₂R^(a), SO₂NHR^(a), NCOR^(a), C(═O)R^(a), CONR²R³,halogen, trihalomethyl, CN, S═O, or nitro; V can be CR²R³, C(═O),C(═O)CR²R³, or C(═N)R²; and W can be O or S. In some instances, V can beC═O, and R¹ can be an optionally substituted aryl, or an optionallysubstituted heteroaryl. For example, R¹ can be optionally substitutedphenyl, or R¹ can be optionally substituted naphthyl.

A class of compounds of interest can include compounds of Formula 1A,1B, or 1C wherein R⁴ can be H; and V can be C═O. Some embodiments caninclude compounds having Formula 1A, Formula, 1B, or Formula 1C whereinR¹ is optionally substituted phenyl or optionally substituted naphthyl.Various embodiments can include compounds having Formula 1A, Formula,1B, or Formula 1C wherein W is S and X is N. Additional embodiments caninclude compounds having Formula 1A, Formula, 1B, or Formula 1C whereinW is O and X is N.

The disclosure also includes a class of compounds represented by any oneof Formulas 2-11.

Formula 12

Embodiments can include compounds having Formula 4 wherein R¹ can be aphenyl group substituted by at least one halogen, a phenyl groupsubstituted by NR²R³, a phenyl group substituted by SO₂NR²R³, CR²R³ORd,an unsubstituted naphthyl group, a naphthyl group substituted byO(CR²R³)_(u)R^(d), NR^(a)(CR²R³)_(u)R^(d), NR^(a)(CR²R³)_(u)NR²R³, a twomembered ring structure including a pyridynyl group and a phenyl group,or a two membered ring structure including a phenyl group and adioxolanyl group; each R^(a) can independently be H or optionallysubstituted hydrocarbyl (C₁-C₁₀); R² and R³ can each independently beR^(a), COR^(a), (CH₂)_(n)O, or SO₂R_(a); each R⁴ can independently beR^(a); R^(d) can be phenyl or morpholino; R⁵ can be H or CH₃; R⁶ can beH or CH₃; and wherein n can be 1, 2, 3, or 4.

Particular embodiments having Formula 4 can be represented by thecompounds

Embodiments can include compounds having Formula 5 wherein R¹ can be aphenyl group substituted by at least one halogen, a phenyl groupsubstituted by NR²R³, a phenyl group substituted by SO₂R^(d), a naphthylgroup optionally substituted by O(CR²R³)_(n)R^(d), or an unsubstitutednaphthyl group, each R^(a) can independently be H or optionallysubstituted C₁-C₁₀ hydrocarbyl; R², R³ and each R⁴ can independently beR^(a), R^(d) can optionally be substituted phenyl or optionallysubstituted morpholino; R⁵ can be H or CH₃; R⁶ can be H or CH₃, andwherein n can be 1, 2, 3, or 4.

Particular embodiments having Formula 4 can be represented by thecompounds

In various embodiments, a compound can be represented by the formula

wherein R⁴ can be R^(d), SO₂R^(d), C(═O)R^(d), NH C(═O)R^(d), R^(e),OR^(c), or CF₃, wherein R^(c) can be H or C₁-C₁₀ hydrocarbyl, R^(d) canbe substituted heterocyclic, unsubstituted heterocyclic, orunsubstituted carbocyclic, and R^(e) can be substituted heteroaryl orsubstituted phenyl; and n can be 1 or 2. In particular embodiments, R⁴can be CF₃, OR^(c), or a phenyl group substituted by at least one OCH₃group.

Additionally, some embodiments of Formula 1D can include compoundsrepresented by

wherein R⁴ can be: (i) C(═O)R^(d) and R^(d) is a pyrrolidonyl group,(ii) SO₂R^(d) and R^(d) is a piperidinyl group, (iii) NHC(═O)R^(d) andR^(d) is a phenyl group or a furanyl group, (iv) an imidazolyl group, or(v) a thiazole group.

Further, some embodiments of Formula 1D can include compoundsrepresented by

wherein X can be NH or O.

Embodiments of Formula 1D can include compounds represented by

In an embodiment, compounds can be represented by the formula

wherein each R⁴ can independently be R², OR^(a), NR²R³, SR^(a), SOR^(a),SO₂R^(a), SO₂NHR^(a), NCOR^(a), C(═O)R^(a), CONR²R³, halogen,trihalomethyl, CN, S═O, or nitro and n=1-4. In some embodiments, R⁴ canbe optionally substituted heteroaryl. Additionally, R⁴ can be optionallysubstituted heteroaryl and n can be 1. In various embodiments, compoundscan be represented by the formula

With respect to any relevant structural feature herein, each R^(a) canindependently be H; optionally substituted hydrocarbyl, such as C₁₋₁₂ orC₁₋₆ hydrocarbyl; optionally substituted aryl, such as optionallysubstituted C₆₋₁₂ aryl, including optionally substituted phenyl;optionally substituted heteroaryl, including optionally substitutedC₂₋₁₂ heteroaryl, such as optionally substituted pyridinyl, optionallysubstituted furyl, optionally substituted thienyl, etc. In someembodiments, each Ra can independently be H, or C₁₋₁₂ alkyl, including:linear or branched alkyl having the formula C_(a)H_(a+1), or cycloalkylhaving the formula C_(a)Ha−1, wherein a is 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, or 12, such as linear or branched alkyl of the formula: CH₃,C₂H₅, C₃H₇, C₄H₉, C₅H₁₁, C₆H₁₃, C₇H₁₅, C₈H₁₇, C₉H₁₉, C_(1o)H₂₁, etc., orcycloalkyl of the formula: C₃H₅, C₄H₇, C₅H₉, C₆H₁₁, C₇H₁₃, C₈H₁₅, C₉H₁₇,C₁₀H₁₉, etc.

With respect to R^(a), in some embodiments, the aryl group can besubstituted with halogen, trihalomethyl, alkoxy, alkylamino, OH, CN,alkylthio, arylthio, sulfoxide, arylsulfonyl, alkylsulfonyl, carboxylicacid, nitro or acylamino.

With respect to R^(a), in some embodiments, the heteroaryl group can besingle or fused. In some embodiments, the single heteroaryl group can beimidazole. In some embodiments, the fused heteroaryl group can bebenzimidazole. In some embodiments, the heteroaryl group can besubstituted with halogen, trihalomethyl, alkoxy, alkylamino, OH, CN,alkylthio, arylthio, sulfoxide, arylsulfonyl, alkylsulfonyl, carboxylicacid, nitro or acylamino. In some embodiments, the alkyl group can bebranched, cyclic or polycyclic.

With respect to R^(a), a hydrocarbyl can be alkyl, alkenyl, or alkynyl.In some embodiments, the alkyl group can be substituted with halogen,trihalomethyl, alkoxy, alkylamino, OH, CN, heteroaryl, alkylthio,arylthio, sulfoxide, arylsulfonyl, alkylsulfonyl, carboxylic acid,nitro, or acylamino. In some embodiments, the heteroaryl group can besingle or fused. In some embodiments, the single heteroaryl group can beimidazole. In some embodiments, the fused heteroaryl group can bebenzimidazole. In some embodiments, the alkenyl group can be branched,cyclic or polycyclic. In some embodiments, the alkenyl group can besubstituted with halogen, trihalomethyl, alkoxy, alkylamino, OH, CN,heteroaryl, alkylthio, arylthio, sulfoxide, arylsulfonyl, alkylsulfonyl,carboxylic acid, nitro, or acylamino.

With respect to any relevant structural feature herein, R^(b) can be H,or C₁₋₃ hydrocarbyl, such as CH₃, C₂H₅, C₃H₇, cyclopropyl, CH═CH₂,CH₂CH═CH₂, C≡CH, CH₂C≡CH, etc.

With respect to any relevant structural feature herein, R^(c) can be H,or C₁₋₃ alkyl, such as CH₃, C₂H₅, C₃H₇, cyclopropyl, etc. In someembodiments, R^(c) can be H.

With respect to any relevant formula or structural depiction herein,such as Formula 1, Formula 2, Formula 3, or Formula 4, R¹ can be R^(a),OR² or NR²R³. In some embodiments, R¹ can be optionally substitutedphenyl. In some embodiments, R¹ can be unsubstituted phenyl. In someembodiments, R¹ can be optionally substituted naphthyl. In someembodiments, R¹ can be unsubstituted naphthyl. In other embodiments, R1can be

In some embodiments, R¹ can be.

In some embodiments, R¹ can be

In some embodiments, R¹ can be

In some embodiments, R¹ can be

In some embodiments, R¹ can be

In some embodiments, R¹ can be

With respect to any relevant structural feature herein, R² can be R^(a),COR^(a), or SO₂R^(a). In some embodiments, R² can be H, methyl, ethyl, apropyl (e.g. n-propyl, isopropyl, etc.), cyclopropyl, a butyl,cyclobutyl or an isomer thereof, a pentyl, cyclopentyl or an isomerthereof, a hexyl, a cyclohexyl or an isomer thereof, etc. In someembodiments, R² can be H.

With respect to any relevant structural feature herein, R³ can be R^(a),C(═O)R^(a), or SO₂R^(a). In some embodiments, R³ can be H, methyl,ethyl, a propyl (e.g. n-propyl, isopropyl, etc.), cyclopropyl, a butyl,cyclobutyl or an isomer thereof, a pentyl, cyclopentyl or an isomerthereof, a hexyl, a cyclohexyl or an isomer thereof, etc. In someembodiments, R³ can be H.

With respect to any relevant structural feature herein, each R⁴ canindependently be R², OR^(a), C(═O)R^(a), CO₂R^(a), OCOR^(a), CONR²R³,NR²R³, NR^(b)C(═O)R^(a), SR^(a), SOR^(a), SO₂R^(a), SO₂NHR^(a),SO₂NR^(a)R^(b), NC(═O)R^(a), halogen, trihalomethyl, CN, S═O, nitro, orC₂₋₅ heteroaryl. In some embodiments, R⁴ can be H.

Generally R⁵ and R⁷-R²², can be H or any substituent, such as asubstituent having from 0 to 6 carbon atoms and from 0 to 5 heteroatomsindependently selected from: O, N, S, F, Cl, Br, and I, and/or having amolecular weight of 15 g/mol to 300 g/mol. Any of R⁵ and R⁷-R²² caninclude: a) 1 or more alkyl moieties optionally substituted with, oroptionally connected by, b) 1 or more functional groups, such as C═C,C≡C, CO, CO₂, CON, NCO₂, OH, SH, O, S, N, N═C, F, Cl, Br, I, CN, NO₂,CO₂H, NH₂, etc.; or can be a substituent having no alkyl portion, suchas F, Cl, Br, I, NO₂, CN, NH₂, OH, COH, CO₂H, etc.

With respect to any relevant structural feature herein, R⁵ can be R^(a),COR^(a), SO₂R^(a), or may not be present. Some examples of R⁵ caninclude H or C₁₋₃ alkyl, such as CH₃, C₂H₅, C₇, cyclopropyl, etc. Insome embodiments, R⁵ can be CH₃ In some embodiments, R⁵ is H.

With respect to any relevant formula or structural depiction above, someexamples of R¹⁹ can include R^(b), OR^(b), SR^(b), C(═O)R^(b), CO₂R^(b),OC(═)R^(b), NR^(b)R^(c), C(═O)NR^(b)R^(c), NR^(b)C(═O)^(c),SO₂NR^(b)R^(c), CF₃, CN, NO₂, F, Cl, Br, I, or C₂₋₅ heterocyclyl. Insome embodiments, R¹⁹ can be H, CH₃, CH₂CH₃, Cl, Br, OH, OCH₃, SCH₃,NH₂, NHCH₃, N(CH₃)₂, SO₂NH₂, morpholino, CH₂C≡CH, or NO₂ In someembodiments, R¹⁹ can be H.

With respect to any relevant formula or structural depiction above, someexamples of R⁵ can include R^(b), or as depicted below, C(═O)R^(b),CO₂R^(b), C(═O)NR^(b)R^(c), NR^(b)C(═O)R^(c), SO₂NR^(b)R^(c), CF₃, CN,or C₂₋₅ heterocyclyl. In some embodiments, R⁵ can be H, CH₃, CH₂CH₃,SO₂NH₂, or CH₂C≡CH. In some embodiments, R⁵ can be H, CH₃, CH₂CH₃,CH₂CH₂CH₃, CH₂CH═CH₂, or CH₂C≡CH. In some embodiments, R⁵ is CH₂C≡CH. Insome embodiments, R⁵ can be H.

With respect to any relevant formula or structural depiction above, someexamples of R⁷ can include R^(b), OR^(b), SR^(b), C(═O)R^(b), CO₂R^(b),OC(═O)R^(b), NR^(b)R^(c), C(═O)NR^(b)R^(c), NR^(b)C(═O)R^(c),SO₂NR^(b)R^(c), CF₃, CN, NO₂, F, Cl, Br, I, or C₂₋₅ heterocyclyl. Insome embodiments, R⁷ can be H, CH₃, CH₂CH₃, Cl, Br, OH, OCH₃, SCH₃, NH₂,NHCH₃, N(CH₃)₂, SO₂NH₂, morpholino, CH₂C≡CH, or NO₂. In someembodiments, R⁷ can be H.

With respect to any relevant formula or structural depiction above, someexamples of R⁸ can include R^(b), OR^(b), SR^(b), C(═O)R^(b), CO₂R^(b),OC(═O)NR^(b), NR^(b)R^(c), C(═)NR^(b)R^(c), NR^(b)C(═O)R^(c),SO₂NR^(b)R^(c), CF₃, CN, NO₂, F, Cl, Br, I, or C₂₋₅ heterocyclyl. Insome embodiments, R⁸ can be H, CH₃, CH₂CH₃, Cl, Br, OH, OCH₃, SCH₃, NH₂,NHCH₃, N(CH₃)₂, SO₂NH₂, morpholino, CH₂C≡CH, or NO₂. In someembodiments, R⁸ can be H, Cl or Br. In some embodiments, R⁸ can be Cl.In some embodiments, R⁸ can be Br. In some embodiments, R⁸ can be H.

With respect to any relevant formula or structural depiction above, someexamples of R⁹ can include R^(b), OR^(b), SR^(b), C(═O)R^(b), CO₂R^(b),OC(═O)NR^(b), NR^(b)R^(c), C(═)NR^(b)R^(c), NR^(b)C(═O)R^(c),SO₂NR^(b)R^(c), CF₃, CN, NO₂, F, Cl, Br, I, or C₂₋₅ heterocyclyl. Insome embodiments, R₉ can be H, CH₃, CH₂CH₃, Cl, Br, OH, OCH₃, SCH₃, NH₂,NHCH₃, N(CH₃)₂, SO₂NH₂, morpholino, CH₂C≡CH, or NO₂. In someembodiments, R⁹ can be H, Cl, or SO₂NH₂. In some embodiments, R⁹ can beH. In some embodiments, R⁹ can be Cl. In some embodiments, R⁹ can beSO₂NH₂. In some embodiments, R⁹ can be H.

In some embodiments, R⁸ and R⁹ can be joined together to form:

With respect to any relevant formula or structural depiction above, someexamples of R¹⁰ can include R^(b), OR^(b), SR^(b), C(═O)R^(b), CO₂R^(b),OC(═O)NR^(b), NR^(b)R^(c), C(═)NR^(b)R^(c), NR^(b)C(═O)R^(c),SO₂NR^(b)R^(c), CF₃, CN, NO₂, F, Cl, Br, I, or C₂₋₅ heterocyclyl. Insome embodiments, R¹⁰ can be H, CH₃, CH₂CH₃, Cl, Br, OH, OCH₃, SCH₃,NH₂, NHCH₃, N(CH₃)₂, SO₂NH₂, morpholino, CH₂C≡CH, or NO₂. In someembodiments, R¹⁰ can be H or Cl. In some embodiments, R¹⁰ can be H. Insome embodiments, R¹⁰ can be Cl. In some embodiments, R⁸ and R¹⁰ can beCl.

With respect to any relevant formula or structural depiction above, someexamples of R¹¹ can include R^(b), OR^(b), SR^(b), C(═O)R^(b), CO₂R^(b),OC(═O)NR^(b), NR^(b)R^(c), C(═)NR^(b)R^(c), NR^(b)C(═O)R^(c), CF₃, CN,NO₂, F, Cl, Br, or I. In some embodiments, R¹¹ can be H, CH₃, CH₂CH₃,Cl, Br, OH, OCH₃, SCH₃, NH₂, NHCH₃, N(CH₃)₂, CH₂C≡CH, or NO₂. In someembodiments, R¹¹ can be H.

In some embodiments, R⁷ and R¹¹ can be H. In some embodiments, R⁷, R⁹,and R¹¹ can be H. In some embodiments, R⁷, R¹⁰, and R¹¹ can be H. Insome embodiments, R⁷, R⁸, R¹⁰, and R¹¹ can be H. In some embodiments,R⁷, R⁸, R⁹, and R¹¹ can be H. In some embodiments, R⁷, R⁸, R⁹, R¹⁰, andR¹¹ can be H.

With respect to any relevant formula or structural depiction above, someexamples of R¹⁵ can include R^(b), OR^(b), SR^(b), C(═O)R^(b), CO₂R^(b),OC(═O)NR^(b), NR^(b)R^(c), C(═)NR^(b)R^(c), NR^(b)C(═O)R^(c),SO2NR^(b)R^(c), CF₃, CN, NO₂, F, Cl, Br, or C₂₋₅ heterocyclyl. In someembodiments, R¹⁵ can be H, CH₃, CH₂CH₃, Cl, Br, OH, OCH₃, SCH₃, NH₂,NHCH₃, N(CH₃)₂, SO₂NH₂, morpholino, CH₂C≡CH, or NO₂. In someembodiments, R¹⁵ can be H.

With respect to any relevant formula or structural depiction above, someexamples of R¹⁶ can include R^(b), OR^(b), SR^(b), C(═O)R^(b), CO2R^(b),OC(═O)R^(b), NR^(b)R^(c), C(═O)NR^(b)R^(c), NR^(b)C(═O)Rc,SO₂NR^(b)R^(c), CF₃, CN, NO₂, F, Cl, Br, I, or C₂₋₅ heterocyclyl. Insome embodiments, R¹⁶ can be H, CH₃, CH₂CH₃, Cl, Br, OH, OCH₃, SCH₃,NH₂, NHCH₃, N(CH₃)₂, SO₂NH₂, CH₂C≡CH, or NO₂. In some embodiments, R¹⁶can be H.

With respect to any relevant formula or structural depiction above, someexamples of R¹⁷ can include R^(b), OR^(b), SR^(b), C(═O)R^(b), CO₂R^(b),OC(═O)R^(b), NR^(b)R^(c), C(═O)NR^(b)R^(c), NR^(b)C(═O)R^(c),SO₂NR^(b)R^(c), CF₃, CN, NO₂, F, Cl, Br, I, or C₂₋₅ heterocyclyl. Insome embodiments, R¹⁷ can be H, CH₃, Cl, Br, OH, OCH₃, SCH₃, NH₂, NHCH₃,N(CH₃)₂, SO₂NH₂, morpholino, CH₂C≡CH, or NO₂. In some embodiments, R¹⁷can be H.

With respect to any relevant formula or structural depiction above, someexamples of R¹⁸ can include R^(b), OR^(b), SR^(b), C(═O)R^(b), CO₂R^(b),OC(═O)R^(b), NR^(b)R^(c), C(═O)NR^(b)R^(c), NR^(b)C(═O)R^(c),SO₂NR^(b)R^(c), CF₃, CN, NO₂, F, Cl, Br, I, or C₂₋₅ heterocyclyl. Insome embodiments, R¹⁸ can be H, CH₃, CH₂CH₃, Cl, Br, OH, OCH₃, SCH₃,NH₂, NHCH₃, N(CH₃)₂, SO₂NH₂, morpholino, or NO₂. In some embodiments,R¹⁸ can be H.

In some embodiments, R¹⁵, R¹⁶, R¹⁷, and R¹⁸ can be H.

With respect to any relevant formula or structural depiction above, someexamples of R¹² can include R^(b), OR^(b), SR^(b), C(═O)R^(b), CO₂R^(b),OC(═O)R^(b), NR^(b)R^(c), C(═O)NR^(b)R^(c), NR^(b)C(═O)R^(c),SO₂NR^(b)R^(c), CF₃, CN, NO₂, F, Cl, Br, I, or C₂₋₅ heterocyclyl. Insome embodiments, R¹² can be H, CH₃, CH₂CH₃, Cl, Br, OH, OCH₃, SCH₃,NH₂, NHCH₃, N(CH₃)₂, SO₂NH₂, CH₂C≡CH, or NO₂. In some embodiments, R12can be H or SO₂NH₂. In some embodiments, R12 can be H. In someembodiments, R12 can be SO₂NH₂.

With respect to any relevant formula or structural depiction above, someexamples of R¹³ can include R^(b), OR^(b), SR^(b), C(═O)R^(b), CO₂R^(b),OC(═O)R^(b), NR^(b)R^(c), C(═O)NR^(b)R^(c), NR^(b)C(═O)R^(c),SO₂NR^(b)R^(c), CF₃, CN, NO₂, F, Cl, Br, I, or C₂₋₅ heterocyclyl. Insome embodiments, R₁₃ can be H, CH₃, CH₂CH₃, Cl, Br, OH, OCH₃, SCH₃,NH₂, NHCH₃, N(CH₃)₂, SO₂NH₂, morpholino, or NO₂. In some embodiments,R¹³ can be H.

In some embodiments, R¹² and R¹³ can be H.

With respect to any relevant formula or structural depiction above, someexamples of R²⁰ can include R^(b), OR^(b), SR^(b), C(═O)R^(b), CO²R^(b),OC(═O)R^(b), NR^(b)R^(c), C(═O)NR^(b)R^(c), NR^(b)C(═O)R^(c),SO²NR^(b)R^(c), CF³, CN, NO², F, Cl, Br, I, or C²⁻⁵ heterocyclyl. Insome embodiments, R²⁰ can be H, CH³, CH²CH³, Cl, Br, OH, OCH³, SCH³,NH², NHCH³, N(CH³)², SO²NH², morpholino, CH²C≡CH, or NO². In someembodiments, R²⁰ can be H, CH²CH³, OCH³, N(CH³)², morpholino, or SCH³.In some embodiments, R²⁰ can be H. In some embodiments, R²⁰ can beCH²CH³. In some embodiments, R²⁰ can be OCH³. In some embodiments, R²⁰can be CN(CH³)². In some embodiments, R²⁰ can be morpholino. In someembodiments, R²⁰ can be SCH³.

With respect to any relevant formula or structural depiction above, someexamples of R22 can include Rb, ORb, SRb, CF3, CN, NO2, F, Cl, Br, I, orC2-5 heterocyclyl. In some embodiments, R22 can be H, CH3, or CH2CH3. Insome embodiments, R22 can be H.

In some embodiments, R¹⁹ and R²² can be H.

With respect to any relevant formula or structural depiction above, someexamples of R¹⁴ can include R^(b), OR^(b), SR^(b), C(═O)R^(b), CO₂R^(b),OC(═O)R^(b), NR^(b)R^(c), C(═O)NR^(b)R^(c), NR^(b)C(═O)R^(c),SO₂NR^(b)R^(c), CF₃, CN, NO₂, F, Cl, Br, or I. In some embodiments, R³⁰can be H, CH₃, CH₂CH₃, Cl, Br, OH, OCH₃, SCH₃, NH₂, NHCH₃, N(CH₃)₂,SO₂NH₂, CH₂C≡CH, or NO₂. In some embodiments, R¹⁴ can be H.

With respect to any relevant formula or structural depiction above,examples of R¹³ can include R^(b), OR^(b), SR^(b), C(═O)R^(b), CO₂R^(b),OC(═O)R^(b), NR^(b)R^(c), C(═O)NR^(b)R^(c), NR^(b)C(═O)R^(c),SO₂NR^(b)R^(c), CF₃, CN, NO₂, F, Cl, Br, I, or C₂₋₅ heterocyclyl. Insome embodiments, R¹³ can be H, CH₃, CH₂CH₃, Cl, Br, OH, OCH₃, SCH₃,NH₂, NHCH₃, N(CH₃)₂, SO₂NH₂, morpholino, CH₂C≡CH, or NO₂. In someembodiments, R¹³ can be H.

With respect to any relevant formula or structural depiction above, someexamples of R¹² can include R^(b), OR^(b), SR^(b), C(═O)R^(b), CO₂R^(b),OC(═O)R^(b), NR^(b)R^(c), C(═O)NR^(b)R^(c), NR^(b)C(═O)R^(c),SO₂NR^(b)R^(c), CF₃, CN, NO₂, F, Cl, Br, I, or C₂₋₅ heterocyclyl. Insome embodiments, R¹² can be H, CH₃, CH₂CH₃, Cl, Br, OH, OCH₃, SCH₃,NH₂, NHCH₃, N(CH₃)₂, SO₂NH₂, morpholino, CH₂C≡CH, or NO₂. In someembodiments, R¹² can be H or NO₂. In some embodiments, R¹² can be H. Insome embodiments, R¹² can be NO₂.

In some embodiments, R¹⁴, R¹³, and R¹² can be H. In some embodiments,R¹³ and R¹² can be H.

As suggested by the list of compounds in Table 1, in some instances, thesubstituent that is joined to an aromatic carbon atom is H.

Specific embodiments of the compounds disclosed herein have thestructures shown in Table 1.

TABLE 1 Structure Compound ID

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

Unless stereochemistry is unambiguously depicted, any structure,formula, or name for a compound can refer to any stereoisomer or anymixture of stereoisomers of the compound.

Unless otherwise indicated, any reference to a compound herein bystructure, formula, name or any other means, includes pharmaceuticallyacceptable salts, such as sodium, potassium, and ammonium salts;prodrugs, such as ester prodrugs; alternate solid forms, such aspolymorphs, solvates, hydrates, etc.; tautomers; isomers; or, any otherchemical species that may rapidly convert to a compound described hereinunder conditions in which the compounds are used as described herein.

As used herein, the term “pharmaceutically acceptable salt” refers topharmaceutical salts that are, within the scope of sound medicaljudgment, suitable for use in contact with the tissues of subjectswithout undue toxicity, irritation, and allergic response, and arecommensurate with a reasonable benefit/risk ratio. Pharmaceuticallyacceptable salts are well known in the art. In one embodiment, thepharmaceutically acceptable salt is a sulfate salt. For example, S. M.Berge, et al. describes pharmaceutically acceptable salts in J. Pharm.Sci., 1977, 66:1-19.

Suitable pharmaceutically acceptable acid addition salts can be preparedfrom an inorganic acid or an organic acid. Examples of such inorganicacids are hydrochloric, hydrobromic, hydroiodic, nitric, carbonic,sulfuric and phosphoric acid. Appropriate organic acids can be selectedfrom aliphatic, cycloaliphatic, aromatic, arylaliphatic, heterocyclic,carboxylic and sulfonic classes of organic acids, examples of which areformic, acetic, propionic, succinic, glycolic, gluconic, maleic, embonic(pamoic), methanesulfonic, ethanesulfonic, 2-hydroxyethanesulfonic,pantothenic, benzenesulfonic, toluenesulfonic, sulfanilic, mesylic,cyclohexylaminosulfonic, stearic, algenic, β-hydroxybutyric, malonic,galactic, and galacturonic acid. Pharmaceutically acceptableacidic/anionic salts also include, the acetate, benzenesulfonate,benzoate, bicarbonate, bitartrate, bromide, calcium edetate, camsylate,carbonate, chloride, citrate, dihydrochloride, edetate, edisylate,estolate, esylate, fumarate, glyceptate, gluconate, glutamate,glycollylarsanilate, hexylresorcinate, hydrobromide, hydrochloride,hydroxynaphthoate, iodide, isethionate, lactate, lactobionate, malate,maleate, malonate, mandelate, mesylate, methylsulfate, mucate,napsylate, nitrate, pamoate, pantothenate, phosphate/diphospate,polygalacturonate, salicylate, stearate, subacetate, succinate, sulfate,hydrogensulfate, tannate, tartrate, teoclate, tosylate, and triethiodidesalts.

Suitable pharmaceutically acceptable base addition salts includemetallic salts made from aluminum, calcium, lithium, magnesium,potassium, sodium, and zinc or organic salts made fromN,N′-dibenzylethylene-diamine, chloroprocaine, choline, diethanolamine,ethylenediamine, N-methylglucamine, lysine, arginine, and procaine. Allof these salts can be prepared by conventional means from thecorresponding compound represented by the disclosed compounds bytreating, for example, the disclosed compounds with the appropriate acidor base. Pharmaceutically acceptable basic/cationic salts also include,the diethanolamine, ammonium, ethanolamine, piperazine, andtriethanolamine salts.

A pharmaceutically acceptable salt includes any salt that retains theactivity of the parent compound and is acceptable for pharmaceuticaluse. A pharmaceutically acceptable salt also refers to any salt whichmay form in vivo as a result of administration of an acid, another salt,or a prodrug which is converted into an acid or salt.

A prodrug includes a compound which is converted to a therapeuticallyactive compound after administration, such as by hydrolysis of an estergroup or some other biologically labile group.

“Functional group” refers to an atom or a group of atoms that havesimilar chemical properties whenever they occur in different compounds,and as such the functional group defines the characteristic physical andchemical properties of families of organic compounds.

Unless otherwise indicated, when any compound or chemical structuralfeature (collectively referred to herein as a “compound”), such as forexample alkyl, aryl, etc., is referred to as being “optionallysubstituted,” that compound can have no substituents (in which case itis “unsubstituted”), or it can include one or more substituents (inwhich case it is “substituted”). The term “substituent” has the ordinarymeaning known to one of ordinary skill in the art. In some embodiments,the substituent can be an ordinary organic moiety known in the art,which can have a molecular weight (e.g., the sum of the atomic masses ofthe atoms of the substituent) of 15 g/mol to 50 g/mol, 15 g/mol to 100g/mol, 15 g/mol to 150 g/mo1,15 g/mol to 200 g/mol, 15 g/mol to 300g/mol, or 15 g/mol to 500 g/mol. In some embodiments, the substituentincludes: 0-30, 0-20, 0-10, or 0-5 carbon (C) atoms; and/or 0-30, 0-20,0-10, or 0-5 heteroatoms including N, O, S, Si, F, Cl, Br, or I;provided that the substituent includes at least one atom including C, N,O, S, Si, F, Cl, Br, or I in a substituted compound. Examples ofsubstituents can include alkyl, alkenyl, alkynyl, heteroalkyl,heteroalkenyl, heteroalkynyl, aryl, heteroaryl, hydroxy, alkoxy,aryloxy, acyl, acyloxy, alkylcarboxylate, thiol, alkylthio, cyano, halo,thiocarbonyl, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl,C-amido, N-amido, S-sulfonamido, N-sulfonamido, isocyanato, thiocyanato,isothiocyanato, nitro, silyl, sulfenyl, sulfinyl, sulfonyl, haloalkyl,haloalkoxyl, trihalomethanesulfonyl, trihalomethanesulfonamido, amino,etc. For convenience, the term “molecular weight” is used with respectto a moiety or part of a molecule to indicate the sum of the atomicmasses of the atoms in the moiety or part of a molecule, even though itmay not be a complete molecule.

“Hydrocarbyl” has the broadest meaning generally understood in the art,and can include a moiety composed of carbon and hydrogen. Some examplescan include alkyl, alkenyl, alkynyl, aryl, etc., and combinationsthereof, and can be linear, branched, cyclic, or a combination thereof.Hydrocarbyl can be bonded to any other number of moieties (for example,can be bonded to one other group, such as —CH₃, —CH═CH₂, etc.; two othergroups, such as -phenyl-, —C≡C—, etc.; or any number of other groups)that the structure can bear, and in some embodiments, can contain fromone to thirty-five carbon atoms. Examples of hydrocarbyl groups includeC1 alkyl, C2 alkyl, C2 alkenyl, C2 alkynyl, C3 alkyl, C3 alkenyl, C3alkynyl, C4 alkyl, C4 alkenyl, C4 alkynyl, C5 alkyl, C5 alkenyl, C5alkynyl, C6 alkyl, C6 alkenyl, C6 alkynyl, phenyl, etc.

“Alkyl” has the broadest meaning generally understood in the art, andcan include a moiety composed of carbon and hydrogen containing nodouble or triple bonds and not having any cyclic structure. Alkyl can belinear alkyl, branched alkyl, cycloalkyl, or a combination thereof, andin some embodiments, can contain from one to thirty-five carbon atoms.In some embodiments, alkyl can include C₁₋₁₀ linear alkyl, such asmethyl (—CH₃), ethyl (—CH₂CH₃), n-propyl (—CH₂CH₂CH₃), n-butyl(—CH₂CH₂CH₂CH₃), n-pentyl (—CH₂CH₂CH₂CH₂CH₃), n-hexyl(—CH₂CH₂CH₂CH₂CH₂CH₃), etc.; C₃₋₁₀ branched alkyl, such as C₃H₇ (e.g.iso-propyl), C₄H₉ (e.g., branched butyl isomers), C₅H₁₁ (e.g., branchedpentyl isomers), C₆H₁₃ (e.g., branched hexyl isomers), C₇H₁₅ (e.g.,branched heptyl isomers), etc.; C₃₋₁₀ cycloalkyl, such as C₃H₅ (e.g.cyclopropyl), C₄H₇ (e.g., cyclobutyl isomers such as cyclobutyl,methylcyclopropyl, etc.), C₅H₉ (e.g., cyclopentyl isomers such ascyclopentyl, methylcyclobutyl, dimethylcyclopropyl, etc.) C₆H₁₁ (e.g.,cyclohexyl isomers), C₇H₁₃ (e.g., cycloheptyl isomers), etc.; C₃₋₁₂bicycloalkyl such as decahydronaphthyl, and norbornyl; and the like.

“Alkyl,” “alkenyl” and “alkynyl” refer to substituted and unsubstitutedalkyls, alkenyls and alkynyls, respectively. An alkyl group can beoptionally substituted as defined herein.

Substituted alkyls, alkenyls, and alkynyls refer to alkyls, alkenyls,and alkynyls substituted with one to five substituents including H,alkyl, aryl, alkenyl, alkynyl, arylalkyl, alkoxy, aryloxy, arylalkoxy,alkoxyalkylaryl, alkylamino, arylamino, NH₂, OH, CN, NO₂, OCF₃, CF₃, F,1-amidine, 2-amidine, alkylcarbonyl, morpholinyl, piperidinyl, dioxanyl,pyranyl, heteroaryl, furanyl, thiophenyl, tetrazolo, thiazolyl,isothiazolyl, imidazolyl, thiadiazolyl, thiadiazole S-oxide, thiadiazoleS,S-dioxide, pyrazolo, oxazolyl, isoxazolyl, pyridinyl, pyrimidinyl,quinolinyl, isoquinolinyl, SR, SOR, SO2R, CO2R, COR, CONR′R″, CSNR′R″,and SOnNR′R″. As used herein, R, R′, and R″ can include R groupsdescribed in this disclosure, such as R^(a), R^(b), R^(c), R^(d), R^(e),R², or R³.

Either alone or in combination, “alkynyl” refers to a functional groupincluding a straight-chain or branched-chain hydrocarbon containing from2 to 20 carbon atoms and having one or more carbon-carbon triple bondsand not having any cyclic structure. An alkynyl group may be optionallysubstituted as defined herein. Examples of alkynyl groups includeethynyl, propynyl, hydroxypropynyl, butynyl, butyn-1-yl, butyn-2-yl,3-methylbutyn-1-yl, pentynyl, pentyn-1-yl, hexynyl, hexyn-2-yl,heptynyl, octynyl, nonynyl, decynyl, undecynyl, dodecynyl, tridecynyl,tetradecynyl, pentadecynyl, hexadecynyl, heptadecynyl, octadecynyl,nonadecynyl, eicosynyl, and the like.

“Alkylene”, alone or in combination, refers to a saturated aliphaticgroup derived from a straight or branched chain saturated hydrocarbonattached at two or more positions, such as methylene (—CH₂—). Unlessotherwise specified, “alkyl” can include “alkylene” groups.

Either alone or in combination “alkylcarbonyl” or “alkanoyl” refer to afunctional group including an alkyl group attached to the parentmolecular moiety through a carbonyl group. Examples of alkylcarbonylgroups can include methylcarbonyl, ethylcarbonyl, and the like.

Either alone or in combination, “heteroalkyl” refers to a functionalgroup including a straight-chain, branched-chain, or cyclic hydrocarboncontaining from 1 to 20 atoms linked exclusively by single bonds, whereat least one atom in the chain is a carbon and at least one atom in thechain is O, S, N, or any combination thereof. The heteroalkyl group canbe fully saturated or contain from 1 to 3 degrees of unsaturation. Thenon-carbon atoms can be at any interior position of the heteroalkylgroup, and up to two non-carbon atoms can be consecutive, such as, e.g.,—CH₂—NH—OCH₃. In addition, the non-carbon atoms can optionally beoxidized and the nitrogen can optionally be quaternized. Examples ofheteroalkyl groups can include morpholine, azanorbornane,tetrahydrofuran, and the like.

Either alone or in combination, “alkyloxy” or “alkoxy” refer to afunctional group including an alkyl ether group. Examples of alkoxys caninclude methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, iso-butoxy,sec-butoxy, tert-butoxy, and the like.

Either alone or in combination, “hydroxy” refers to the functional grouphydroxyl (—OH).

Either alone or in combination, “carboxyl” or “carboxy” refers to thefunctional group —C(═O)OH or the corresponding “carboxylate” anion—C(═O)O—. Examples include formic acid, acetic acid, oxalic acid, andbenzoic acid. An “O-carboxyl” group refers to a carboxyl group havingthe general formula RCOO, wherein R is an organic moiety or group. A“C-carboxyl” group refers to a carboxyl group having the general formulaCOOR, wherein R is an organic moiety or group.

Either alone or in combination, “oxo” refers to the functional group ═O.

“Carbocyclic” has the broadest meaning generally understood in the art,and includes a ring or ring system wherein the ring atoms are allcarbon. Examples can include phenyl, naphthyl, anthracenyl, cycloalkyl,cycloalkenyl, cycloalkynyl, etc., and combinations thereof.

“Heterocyclic” has the broadest meaning generally understood in the art,and includes a ring or ring system wherein at least one of the ringatoms is not carbon, such as N, O, S, etc. Examples can includeheteroaryl, cycloheteroalkyl, cycloheteroalkenyl, cycloheteroalkynyl,cyclic heteroalkyl, etc., and combinations thereof. Examples ofheterocyclic systems can include quinoline, tetrahydroisoquinoline,tetrahydropyran, imidazole, thiophene, dihydrobenzofuran, and the like.

Either alone or in combination, “cycloalkyl,” “carbocyclicalkyl” and“carbocyclealkyl” refer to a functional group including a substituted orunsubstituted non-aromatic hydrocarbon with a non-conjugated cyclicmolecular ring structure of 3 to 12 carbon atoms linked exclusively withcarbon-carbon single bonds in the carbon ring structure. A cycloalkylgroup can be monocyclic, bicyclic or polycyclic, and can optionallyinclude one to three additional ring structures, such as, e.g., an aryl,a heteroaryl, a cycloalkenyl, a heterocycloalkyl, or aheterocycloalkenyl.

Either alone or in combination, “lower cycloalkyl” refers to afunctional group including a monocyclic substituted or unsubstitutednon-aromatic hydrocarbon with a non-conjugated cyclic molecular ringstructure of 3 to 6 carbon atoms linked exclusively with carbon-carbonsingle bonds in the carbon ring structure. Examples of lower cycloalkylgroups can include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

“Aryl” has the broadest meaning generally understood in the art, and caninclude an aromatic ring or aromatic ring system. An aryl group can bemonocyclic, bicyclic or polycyclic, and may optionally include one tothree additional ring structures; such as, for example, a cycloalkyl, acycloalkenyl, a heterocycloalkyl, a heterocycloalkenyl, or a heteroaryl.The term “aryl” includes phenyl (benzenyl), thiophenyl, indolyl,naphthyl, tolyl, xylyl, anthracenyl, phenanthryl, azulenyl, biphenyl,naphthalenyl, 1-methylnaphthalenyl, acenaphthenyl, acenaphthylenyl,anthracenyl, fluorenyl, phenalenyl, phenanthrenyl, benzo[a]anthracenyl,benzo[c]phenanthrenyl, chrysenyl, fluoranthenyl, pyrenyl, tetracenyl(naphthacenyl), triphenylenyl, anthanthrenyl, benzopyrenyl,benzo[a]pyrenyl, benzo[e]fluoranthenyl, benzo[ghi]perylenyl,benzo[j]fluoranthenyl, benzo[k]fluoranthenyl, corannulenyl, coronenyl,dicoronylenyl, helicenyl, heptacenyl, hexacenyl, ovalenyl, pentacenyl,picenyl, perylenyl, tetraphenylenyl, etc.

Additionally, either alone or in combination, “aryl,” “hydrocarbyl aryl”or “aryl hydrocarbon” can refer to a functional group including asubstituted or unsubstituted aromatic hydrocarbon with a conjugatedcyclic molecular ring structure of 3 to 12 carbon atoms. Substitutedaryl refers to aryls substituted with one to five substituents includingH, lower alkyl, aryl, alkenyl, alkynyl, arylalkyl, alkoxy, aryloxy,arylalkoxy, alkoxyalkylaryl, alkylamino, arylamino, NH₂, OH, CN, NO₂,OCF₃, CF₃, Br, Cl, F, 1-amidino, 2-amidino, alkylcarbonyl, morpholino,piperidinyl, dioxanyl, pyranyl, heteroaryl, furanyl, thiophenyl,tetrazolo, thiazole, isothiazolo, imidazolo, thiadiazole, thiadiazoleS-oxide, thiadiazole S,S-dioxide, pyrazolo, oxazole, isoxazole,pyridinyl, pyrimidinyl, quinoline, isoquinoline, SR, SOR, SO₂R, CO₂R,COR, CONR′R″, CSNR′R″, SO_(n)NR′R″, etc.

Either alone or in combination, “lower aryl” refers to a functionalgroup including a substituted or unsubstituted aromatic hydrocarbon witha conjugated cyclic molecular ring structure of 3 to 10 carbon atoms.Examples of lower aryl groups can include phenyl and naphthyl.

Either alone or in combination, “heteroaryl” refers to a functionalgroup including a substituted or unsubstituted aromatic hydrocarbon witha conjugated cyclic molecular ring structure of 3 to 12 atoms, where atleast one atom in the ring structure is a carbon and at least one atomin the ring structure is O, S, N, or any combination thereof. Aheteroaryl group can be monocyclic, bicyclic or polycyclic, and mayoptionally include one to three additional ring structures, such as,e.g., an aryl, a cycloalkyl, a cycloalkenyl, a heterocycloalkyl, or aheterocycloalkenyl. Examples of heteroaryl groups can include acridinyl,benzidolyl, benzimidazolyl, benzisoxazolyl, benzodioxinyl,dihydrobenzodioxinyl, benzodioxolyl, 1,3-benzodioxolyl, benzofuryl,benzoisoxazolyl, benzopyranyl, benzothiophenyl, benzo[c]thiophenyl,benzotriazolyl, benzoxadiazolyl, benzoxazolyl, benzothiadiazolyl,benzothiazolyl, benzothienyl, carbazolyl, chromonyl, cinnolinyl,dihydrocinnolinyl, coumarinyl, dibenzofuranyl, furopyridinyl, furyl,indolizinyl, indolyl, dihydroindolyl, imidazolyl, indazolyl,isobenzofuryl, isoindolyl, isoindolinyl, dihydroisoindolyl, isoquinolyl,dihydroisoquinolinyl, isoxazolyl, isothiazolyl, oxazolyl, oxadiazolyl,phenanthrolinyl, phenanthridinyl, purinyl, pyranyl, pyrazinyl,pyrazolyl, pyridyl, pyrimidinyl, pyridazinyl, pyrrolinyl, pyrrolyl,pyrrolopyridinyl, quinolyl, quinoxalinyl, quinazolinyl,tetrahydroquinolinyl, tetrazolopyridazinyl, tetrahydroisoquinolinyl,thiophenyl, thiazolyl, thiadiazolyl, thienopyridinyl, thienyl,thiophenyl, triazolyl, xanthenyl, and the like.

The phenyl structure associated with some of the embodiments describedherein is depicted below. This structure can be unsubstituted, as shownbelow, or can be substituted such that a substituent can independentlybe in any position normally occupied by a hydrogen atom when thestructure is unsubstituted. Unless a point of attachment is indicated by−I, attachment may occur at any position normally occupied by a hydrogenatom.

Each R^(a) can independently be H; optionally substituted hydrocarbyl;optionally substituted aryl, such as optionally substituted phenyl oroptionally substituted aryl; optionally substituted heteroaryl, such asoptionally substituted pyridinyl, optionally substituted furyl,optionally substituted thienyl, etc. In some embodiments, each R^(a) canindependently be H, or C₁₋₁₂ alkyl, including: linear or branched alkylsuch as linear or branched alkyl of the formula: CH₃, C₂H₅, C₃H₇, C₄H₉,C₅H₁₁, C₆H₁₃, C₇H₁₅, C₈H₁₇, C₉H₁₉, C₁₀H₂₁, etc., or cycloalkyl of theformula: C₃H₅, C₄H₇, C₅H₉, C₆H₁₁, C₇H₁₃, C₈H₁₅, C₉H₁₇, C₁₀H₁₉, etc.

Pharmaceutical Compositions

According to other embodiments, the present disclosure provides for apharmaceutical composition including any one of the compounds describedherein.

Pharmaceutical compositions can be formed by combining a compounddisclosed herein, or a pharmaceutically acceptable prodrug or saltthereof, with a pharmaceutically acceptable carrier suitable fordelivery to a subject in accordance with known methods of drug delivery.Accordingly, a “pharmaceutical composition” includes at least onecompound disclosed herein together with one or more pharmaceuticallyacceptable carriers, excipients, or diluents, as appropriate for thechosen mode of administration.

A pharmaceutical composition including a compound of the disclosure canbe formulated in a variety of forms depending upon the particularindication being treated and will be apparent to one of ordinary skillin the art. Formulating pharmaceutical compositions including one ormore compounds of the disclosure can employ straightforward medicinalchemistry processes. The pharmaceutical compositions can be subjected toconventional pharmaceutical operations such as sterilization and/or cancontain conventional adjuvants, such as buffering agents, preservatives,isotonicifiers, stabilizers, wetting agents, emulsifiers, etc.

The administration of the formulations of the present disclosure can beperformed in a variety of ways, including orally, subcutaneously,intravenously, intracerebrally, intranasally, transdermally,intraperitoneally, intramuscularly, intrapulmonary, intrathecally,vaginally, rectally, intraocularly, or in any other acceptable manner.The formulations can be administered continuously by infusion, althoughbolus injection is acceptable, using techniques known in the art, suchas pumps (e.g., subcutaneous osmotic pumps) or implantation. In someinstances the formulations can be directly applied as a solution orspray.

An example of a pharmaceutical composition is a solution designed forparenteral administration. Although in many cases pharmaceuticalsolution formulations are provided in liquid form, appropriate forimmediate use, such parenteral formulations can also be provided infrozen or in lyophilized form. In the former case, the composition mustbe thawed prior to use. The latter form is often used to enhance thestability of the active compound contained in the composition under awider variety of storage conditions, as it is recognized by those ofordinary skill in the art that lyophilized preparations are generallymore stable than their liquid counterparts. Such lyophilizedpreparations are reconstituted prior to use by the addition of one ormore suitable pharmaceutically acceptable diluents such as sterile waterfor injection or sterile physiological saline solution.

Parenterals can be prepared for storage as lyophilized formulations oraqueous solutions by mixing, as appropriate, the compound having thedesired degree of purity with one or more pharmaceutically acceptablecarriers, excipients or stabilizers typically employed in the art (allof which are termed “excipients”), for example buffering agents,stabilizing agents, preservatives, isotonifiers, non-ionic detergents,antioxidants and/or other miscellaneous additives.

Buffering agents help to maintain the pH in the range which approximatesphysiological conditions. They are typically present at a concentrationranging from 2 mM to 50 mM of a pharmaceutical composition. Suitablebuffering agents for use with the present disclosure include bothorganic and inorganic acids and salts thereof, such as citrate buffers(e.g., monosodium citrate-disodium citrate mixture, citricacid-trisodium citrate mixture, citric acid-monosodium citrate mixture,etc.), succinate buffers (e.g., succinic acid-monosodium succinatemixture, succinic acid-sodium hydroxide mixture, succinic acid-disodiumsuccinate mixture, etc.), tartrate buffers (e.g., tartaric acid-sodiumtartrate mixture, tartaric acid-potassium tartrate mixture, tartaricacid-sodium hydroxide mixture, etc.), fumarate buffers (e.g., fumaricacid-monosodium fumarate mixture, fumaric acid-disodium fumaratemixture, monosodium fumarate-disodium fumarate mixture, etc.), gluconatebuffers (e.g., gluconic acid-sodium glyconate mixture, gluconicacid-sodium hydroxide mixture, gluconic acid-potassium glyconatemixture, etc.), oxalate buffer (e.g., oxalic acid-sodium oxalatemixture, oxalic acid-sodium hydroxide mixture, oxalic acid-potassiumoxalate mixture, etc.), lactate buffers (e.g., lactic acid-sodiumlactate mixture, lactic acid-sodium hydroxide mixture, lacticacid-potassium lactate mixture, etc.), and acetate buffers (e.g., aceticacid-sodium acetate mixture, acetic acid-sodium hydroxide mixture,etc.). Additional possibilities are phosphate buffers, histidinebuffers, and trimethylamine salts such as Tris.

Preservatives can be added to retard microbial growth, and are typicallyadded in amounts of 0.2%-1% (w/v). Suitable preservatives for use withthe present disclosure include phenol, benzyl alcohol, meta-cresol,methyl paraben, propyl paraben, octadecyldimethylbenzyl ammoniumchloride, benzalkonium halides (e.g., benzalkonium chloride, bromide oriodide), hexamethonium chloride, alkyl parabens such as methyl or propylparaben, catechol, resorcinol, cyclohexanol and 3-pentanol.

Isotonicifiers can be added to ensure isotonicity of liquid compositionsand include polyhydric sugar alcohols, preferably trihydric or highersugar alcohols, such as glycerin, erythritol, arabitol, xylitol,sorbitol and mannitol. Polyhydric alcohols can be present in an amountbetween 0.1% and 25% by weight, typically 1% to 5%, taking into accountthe relative amounts of the other ingredients.

Stabilizers refer to a broad category of excipients which can range infunction from a bulking agent to an additive which solubilizes thetherapeutic agent or helps to prevent denaturation or adherence to thecontainer wall. Typical stabilizers can be polyhydric sugar alcohols(enumerated above); amino acids such as arginine, lysine, glycine,glutamine, asparagine, histidine, alanine, ornithine, L-leucine,2-phenylalanine, glutamic acid, threonine, etc.; organic sugars or sugaralcohols, such as lactose, trehalose, stachyose, mannitol, sorbitol,xylitol, ribitol, myoinisitol, galactitol, glycerol and the like,including cyclitols such as inositol; polyethylene glycol; amino acidpolymers; sulfur-containing reducing agents, such as urea, glutathione,thioctic acid, sodium thioglycolate, thioglycerol,alpha-monothioglycerol and sodium thiosulfate; low molecular weightpolypeptides (i.e., <10 residues); proteins such as human serum albumin,bovine serum albumin, gelatin or immunoglobulins; hydrophilic polymerssuch as polyvinylpyrrolidone; monosaccharides such as xylose, mannose,fructose and glucose; disaccharides such as lactose, maltose andsucrose; trisaccharides such as raffinose, and polysaccharides such asdextran. Stabilizers are typically present in the range of from 0.1 to10,000 parts by weight based on the active compound weight.

Additional miscellaneous excipients include fillers (e.g., starch),chelating agents (e.g., EDTA), antioxidants (e.g., ascorbic acid,methionine, vitamin E), and cosolvents.

Particular embodiments can include one or more of ethanol (<10%),propylene glycol (<40%), polyethylene glycol (PEG) 300 or 400 (<60%),N-N-dimethylacetamide (DMA, <30%), N-methyl-2-pyrrolidone (NMP, <20%),dimethyl sulfoxide (DMSO, <20%) co-solvents or the cyclodextrins (<40%)and have a pH of 3 to 9.

The compound(s) can also be entrapped in microcapsules prepared, forexample, by coascervation techniques or by interfacial polymerization,for example hydroxymethylcellulose, gelatin or poly-(methylmethacrylate)microcapsules, in colloidal drug delivery systems (for exampleliposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington, The Science and Practice of Pharmacy, 21st Ed., published byLippincott Williams & Wilkins, A Wolters Kluwer Company, 2005.

Parenteral formulations to be used for in vivo administration generallyare sterile. This is readily accomplished, for example, by filtrationthrough sterile filtration membranes.

Generally, the pharmaceutical compositions can be made up in a solidform (including granules, powders, or suppositories) or in a liquid form(e.g., solutions, suspensions, or emulsions). The compounds can beadmixed with adjuvants such as lactose, sucrose, starch powder,cellulose esters of alkanoic acids, stearic acid, talc, magnesiumstearate, magnesium oxide, sodium and calcium salts of phosphoric andsulphuric acids, acacia, gelatin, sodium alginate,polyvinyl-pyrrolidine, and/or polyvinyl alcohol, and tableted orencapsulated for conventional administration. Alternatively, they can bedissolved in saline, water, polyethylene glycol, propylene glycol,ethanol, oils (such as corn oil, peanut oil, cottonseed oil, or sesameoil), tragacanth gum, and/or various buffers. Other adjuvants and modesof administration are well known in the pharmaceutical art. The carrieror diluent can include time delay material, such as glycerylmonostearate or glyceryl distearate alone or with a wax, or othermaterials well known in the art.

Oral administration of the compounds and compositions is one intendedpractice of the disclosure. For oral administration, the pharmaceuticalcomposition can be in solid or liquid form, e.g., in the form of acapsule, tablet, powder, granule, suspension, emulsion or solution. Thepharmaceutical composition is preferably made in the form of a dosageunit containing a given amount of the active ingredient. A suitabledaily dose for a human or other subject can vary widely depending on thecondition of the subject and other factors, but can be determined bypersons of ordinary skill in the art using routine methods.

Solid dosage forms for oral administration can include capsules,tablets, pills, powders, and granules. In such solid dosage forms, theactive compound can be admixed with at least one inert diluent such assucrose, lactose, or starch. Such dosage forms can also include, as isnormal practice, additional substances other than inert diluents, e.g.,lubricating agents such as magnesium stearate. In the case of capsules,tablets, and pills, the dosage forms can also include buffering agents.Tablets and pills can additionally be prepared with enteric coatings.For buccal administration the pharmaceutical compositions can take theform of tablets or lozenges formulated in conventional manners.

Liquid dosage forms for oral administration can include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups, and elixirscontaining inert diluents commonly used in the art, such as water. Suchpharmaceutical compositions can also include adjuvants, such as wetting,sweetening, flavoring, and perfuming agents.

The pharmaceutical compositions can be formulated for parenteraladministration by injection, e.g. by bolus injection, or infusion.Formulations for injection can be presented in unit dosage form, e.g. inglass ampoule or multi-dose containers, e.g. glass vials. Thepharmaceutical compositions for injection can take such forms assuspensions, solutions, or emulsions in oily or aqueous vehicles, andcan contain formulatory agents such as antioxidants, buffers, non-ionicdetergents, dispersants, isotonicifiers, suspending agents, stabilizers,preservatives, dispersing agents and/or other miscellaneous additives.

Although in many cases pharmaceutical compositions provided in liquidform are appropriate for immediate use, such parenteral formulations canalso be provided in frozen or in lyophilized form. In the former case,the pharmaceutical composition must be thawed prior to use. The latterform is often used to enhance the stability of the compound contained inthe pharmaceutical composition under a wider variety of storageconditions, as it is recognized by those or ordinary skill in the artthat lyophilized preparations are generally more stable than theirliquid counterparts. Parenterals can be prepared for storage aslyophilized formulations by mixing, as appropriate, the compound havingthe desired degree of purity with one or more pharmaceuticallyacceptable carriers, excipients, or stabilizers typically employed inthe art (all of which are termed “excipients”), for example,antioxidants, buffers, non-ionic detergents, dispersants,isotonicifiers, suspending agents, stabilizers, preservatives,dispersing agents and/or other miscellaneous additives. Such lyophilizedpreparations are reconstituted prior to use by the addition of one ormore suitable pharmaceutically acceptable diluents such as sterilepyrogen-free water for injection or sterile physiological salinesolution.

For administration by inhalation (e.g., nasal or pulmonary), thepharmaceutical compositions can be conveniently delivered in the form ofan aerosol spray, from pressurized packs or a nebulizer, and/or with theuse of suitable propellant, e.g. dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, orother suitable gases or mixture of gases.

The compounds or compositions can be admixed with adjuvants such aslactose, sucrose, starch powder, cellulose esters of alkanoic acids,stearic acid, talc, magnesium stearate, magnesium oxide, sodium andcalcium salts of phosphoric and sulphuric acids, acacia, gelatin, sodiumalginate, polyvinyl-pyrrolidine, and/or polyvinyl alcohol, and tabletedor encapsulated for conventional administration. Alternatively, they canbe dissolved in saline, water, polyethylene glycol, propylene glycol,ethanol, oils (such as corn oil, peanut oil, cottonseed oil or sesameoil), tragacanth gum, and/or various buffers. Other adjuvants and modesof administration are known in the pharmaceutical art. The carrier ordiluent can include time delay material, such as glyceryl monostearateor glyceryl distearate alone or with a wax, or other materials known inthe art.

In addition to the formulations described above, the pharmaceuticalcompositions can also be formulated as depot preparations. Such longacting formulations can be administered by implantation or byintramuscular injection.

The compounds can also be entrapped in microcapsules prepared, forexample, by coascervation techniques or by interfacial polymerization,(for example hydroxymethylcellulose, gelatin or poly-(methylmethacylate)microcapsules), in colloidal drug delivery systems (for exampleliposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington, The Science and Practice of Pharmacy, 21st Ed., published byLippincott Williams & Wilkins, A Wolters Kluwer Company, 2005.

Suitable examples of sustained-release preparations includesemi-permeable matrices of solid hydrophobic polymers containing thecompound or composition, the matrices having a suitable form such as afilm or microcapsules. Examples of sustained-release matrices includepolyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate) orpoly(vinylalcohol)), polylactides, copolymers of L-glutamic acid andethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the PROLEASE® technology(Alkermes, Cambridge, Massachusetts) or LUPRON DEPOT® (injectablemicrospheres composed of lactic acid-glycolic acid copolymer andleuprolide acetate; Abbott Laboratories, Abbott Park, Ill.), andpoly-D-(−)-3-hydroxybutyric acid. While polymers such as ethylene-vinylacetate and lactic acid-glycolic acid enable release of molecules forlong periods such as up to or over 100 days, certain hydrogels releasecompounds for shorter time periods.

Methods of Use

The pharmaceutical compositions disclosed herein can be used to treat aviral infection in a subject; wherein the viral infection is caused by avirus from one the following families: Arenaviridae, Arterivirus,Astroviridae, Birnaviridae, Bromoviridae, Bunyaviridae, Caliciviridae,Closteroviridae, Comoviridae, Coronaviridae, Cystoviridae, Filoviridae,Flaviviridae, Flexiviridae, Hepadnaviridae, Hepevirus, Herpesviridae,Leviviridae, Luteoviridae, Mesoniviridae, Mononegavirales, MosaicViruses, Nidovirales, Nodaviridae, Orthomyxoviridae, Papillomaviridae,Paramyxoviridae, Picobirnaviridae, Picobirnavirus, Picornaviridae,Potyviridae, Reoviridae, Retroviridae, Roniviridae, Sequiviridae,Tenuivirus, Togaviridae, Tombusviridae, Totiviridae, and Tymoviridae.

According to more specific embodiments, the pharmaceutical compositionscan be used to treat a viral infection caused by one or more of Alfuyvirus, Banzi virus, bovine diarrhea virus, Chikungunya virus, Denguevirus (DNV), Encephalomyocarditis virus (EMCV), Hepatitis B virus (HBV),HCV, human cytomegalovirus (hCMV), HIV, Ilheus virus, influenza virus(including avian and swine isolates), rhinovirus, norovirus, adenovirus,Japanese encephalitis virus, Kokobera virus, Kunjin virus, Kyasanurforest disease virus, louping-ill virus, measles virus, MERS-coronavirus(MERS), metapneumovirus, any of the Mosaic Viruses, Murray Valley virus,parainfluenza virus, poliovirus, Powassan virus, respiratory syncytialvirus (RSV), Rocio virus, SARS-coronavirus (SARS), St. Louisencephalitis virus, tick-borne encephalitis virus, WNV, Ebola virus,Nipah virus, Lassa virus, Tacaribe virus, Junin virus, and yellow fevervirus.

In an embodiment, a method of treating a viral infection in a subjectcan include administering to the subject a therapeutically effectiveamount of a pharmaceutical composition having the structure

In some cases, the viral infection is caused by Ebola virus.

Many RNA viruses share biochemical, regulatory, and signaling pathways.These viruses include influenza viruses (including avian and swineisolates), rhinovirus, norovirus, DNV, RSV, WNV, HCV, parainfluenzavirus, metapneumovirus, Chikungunya virus, SARS, MERS, poliovirus,measles virus, yellow fever virus, tick-borne encephalitis virus,Japanese encephalitis virus, St. Louis encephalitis virus, Murray Valleyvirus, Powassan virus, Rocio virus, louping-ill virus, Banzi virus,Ilheus virus, Kokobera virus, Kunjin virus, Alfuy virus, bovine diarrheavirus, any of the Mosaic Viruses, HIV, Ebola virus, Lassa virus, and theKyasanur forest disease virus. The compounds, pharmaceuticalcompositions, and methods disclosed herein can be used to treat theseviruses.

Antiviral activity against WNV, Nipah Virus, Lassa Fever Virus, andEbola Virus in vitro is measured by focus-forming assay. Virus strainsthat are used in these assays include WNV-TX (WNV), WNV-MAD (WNV),NiV-Malaysia (Nipah), LAS V-Josiah (Lassa Fever), and ZEBOV-Mayinga(Ebola). Cultured human cells including human umbilical vein cells(HUVEC) are seeded in tissue-culture plates and infected with virus atMOI of 0.01 to 0.5 for a duration including but not limited to 2 hoursand then removed. Compound dilutions are prepared in 0.5% DMSO and usedto treat cells at final concentrations of compound ranging 0.001 to 10μM per well. Vehicle control wells contain 0.5% DMSO and are used tocompare to drug treated cells. Virus infections after drug treatment areallowed to proceed for 48 to 96 hours. Virus supernatants are thenharvested and used to infect new monolayer of permissive cells. Thenewly infected cells are incubated overnight (18-24 hours) and used tomeasure the level of infectious virus in the original supernatants byfocus-forming assay using methods generally known in the art.

Methods disclosed herein include treating subjects (humans, mammals,free-range herds, veterinary animals (dogs, cats, reptiles, birds,etc.), farm animals and livestock (horses, cattle, goats, pigs,chickens, etc.), and research animals (monkeys, rats, mice, fish, etc.))with pharmaceutical compositions disclosed herein. Treating subjectsincludes delivering therapeutically effective amounts. Therapeuticallyeffective amounts include those that provide effective amounts,prophylactic treatments, and/or therapeutic treatments.

An “effective amount” is the amount of a compound necessary to result ina desired physiological change in the subject. Effective amounts areoften administered for research purposes. Effective amounts disclosedherein reduce, control, or eliminate the presence or activity of viralinfections and/or reduce, control, or eliminate unwanted side effects ofviral infections. For example, an effective amount may result in areduction in viral protein in a subject or assay, a reduction in viralRNA in a subject or assay, and/or a reduction in virus present in a cellculture.

A “prophylactic treatment” includes a treatment administered to asubject who does not display signs or symptoms of a viral infection ordisplays only early signs or symptoms of the viral infection such thattreatment is administered for the purpose of diminishing, preventing, ordecreasing the risk of developing the viral infection further. Thus, aprophylactic treatment functions as a preventative treatment against aviral infection. Prophylactic treatment may also include vaccines asdescribed elsewhere herein. Prophylactic treatment may result in a lackof increase in viral proteins or RNA in a subject, and/or a lack ofincrease in clinical indicators of viral infection, such as: loss ofappetite, fatigue, fever, muscle aches, nausea, and/or abdominal pain inthe case of HCV; fever and/or headache in the case of WNV; and cough,congestion, fever, sore throat, and/or headache in the case of RSV.Prophylactic treatments can be administered to any subject regardless ofwhether signs of viral infection are present. In some embodiments,prophylactic treatments can be administered before travel.

A “therapeutic treatment” includes a treatment administered to a subjectwho displays symptoms or signs of a viral infection and is administeredto the subject for the purpose of diminishing or eliminating the signsor symptoms of the viral infection. The therapeutic treatment canreduce, control, or eliminate the presence or activity of viruses and/orreduce, control, or eliminate side effects of viruses. Therapeutictreatment may result in a decrease in viral proteins or RNA in asubject, and/or a decrease in clinical indicators of viral infection,such as: loss of appetite, fatigue, fever, muscle aches, nausea, and/orabdominal pain in the case of HCV; fever and/or headache in the case ofWNV; and cough, congestion, fever, cyanosis, sore throat, and/orheadache in the case of RSV.

For administration, therapeutically effective amounts (also referred toherein as doses) can be initially estimated based on results from invitro assays and/or animal model studies. For example, a dose can beformulated in animal models to achieve a circulating concentration rangethat includes an IC50 as determined in cell culture against a particulartarget. Such information can be used to more accurately determine usefuldoses in subjects of interest.

The actual dose amount administered to a particular subject can bedetermined by a physician, veterinarian, or researcher taking intoaccount parameters such as physical and physiological factors includingtarget, body weight, severity of condition, type of viral infection,previous or concurrent therapeutic interventions, idiopathy of thesubject, and route of administration.

Pharmaceutical compositions can be administered intravenously to asubject for treatment of viral infections in a clinically safe andeffective manner, including one or more separate administrations of thecomposition. For example, 0.05 mg/kg to 5.0 mg/kg can be administered toa subject per day in one or more doses (e.g., doses of 0.05 mg/kgonce-daily (QD), 0.10 mg/kg QD, 0.50 mg/kg QD, 1.0 mg/kg QD, 1.5 mg/kgQD, 2.0 mg/kg QD, 2.5 mg/kg QD, 3.0 mg/kg QD, 0.75 mg/kg twice-daily(BID), 1.5 mg/kg BID or 2.0 mg/kg BID). For certain antiviralindications, the total daily dose of a compound can be 0.05 mg/kg to 3.0mg/kg administered intravenously to a subject one to three times a day,including administration of total daily doses of 0.05-3.0, 0.1-3.0,0.5-3.0, 1.0-3.0, 1.5-3.0, 2.0-3.0, 2.5-3.0, and 0.5-3.0 mg/kg/day ofcompounds of Table 1 using 60-minute QD, BID, or three times daily (TID)intravenous infusion dosing. In one particular example, antiviralpharmaceutical compositions can be intravenously administered QD or BIDto a subject with, e.g., total daily doses of 1.5 mg/kg, 3.0 mg/kg, 4.0mg/kg of a composition with up to 92-98% wt/wt of a compound of Table 1.

Additional useful doses can often range from 0.1 to 5 μg/kg or from 0.5to 1 μg/kg. In other examples, a dose can include 1 μg/kg, 5 μg/kg, 10μg/kg, 15 μg/kg, 20 μg/kg, 25 μg/kg, 30 μg/kg, 35 μg/kg, 40 μg/kg, 45μg/kg, 50 μg/kg, 55 μg/kg, 60 μg/kg, 65 μg/kg, 70 μg/kg, 75 μg/kg, 80μg/kg, 85 μg/kg, 90 μg/kg, 95 μg/kg, 100 μg/kg, 150 μg/kg, 200 μg/kg,250 μg/kg, 350 μg/kg, 400 μg/kg, 450 μg/kg, 500 μg/kg, 550 μg/kg, 600μg/kg, 650 μg/kg, 700 μg/kg, 750 μg/kg, 800 μg/kg, 850 μg/kg, 900 μg/kg,950 μg/kg, 1000 μg/kg, 0.1 to 5 mg/kg, or from 0.5 to 1 mg/kg. In otherexamples, a dose can include 1 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 55mg/kg, 60 mg/kg, 65 mg/kg, 70 mg/kg, 75 mg/kg, 80 mg/kg, 85 mg/kg, 90mg/kg, 95 mg/kg, 100 mg/kg, 150 mg/kg, 200 mg/kg, 250 mg/kg, 350 mg/kg,400 mg/kg, 450 mg/kg, 500 mg/kg, 550 mg/kg, 600 mg/kg, 650 mg/kg, 700mg/kg, 750 mg/kg, 800 mg/kg, 850 mg/kg, 900 mg/kg, 950 mg/kg, 1000mg/kg, or more.

Therapeutically effective amounts can be achieved by administeringsingle or multiple doses during the course of a treatment regimen (e.g.,daily, every other day, every 3 days, every 4 days, every 5 days, every6 days, weekly, every 2 weeks, every 3 weeks, monthly, every 2 months,every 3 months, every 4 months, every 5 months, every 6 months, every 7months, every 8 months, every 9 months, every 10 months, every 11months, or yearly.

The administration of the pharmaceutical compositions of the presentdisclosure can be performed in a variety of ways, including orally,subcutaneously, intravenously, intracerebrally, intranasally,transdermally, intraperitoneally, intramuscularly, intrapulmonary,intrathecally, vaginally, rectally, intraocularly, or in any otheracceptable manner. The pharmaceutical compositions can be administeredcontinuously by infusion, although bolus injection is acceptable, usingtechniques well known in the art, such as pumps (e.g., subcutaneousosmotic pumps) or implantation. In some instances the pharmaceuticalcompositions can be directly applied as a solution or spray.

Particular embodiments provide for pharmaceutical compositions includingany one or more of the compounds described herein, for the purpose oftreating and/or preventing disease in a subject. Additional embodimentsprovide for pharmaceutical compositions alone or in combination with anantigen. As such, in some embodiments the pharmaceutical compositionscan be used as vaccines.

The disclosure provides for the use of the compounds as adjuvants.

The compounds, pharmaceutical compositions, and methods disclosed hereincan be additive or synergistic with other therapies currently indevelopment or use. For example, ribavirin and interferon-α provide aneffective treatment for HCV infection when used in combination. Theirefficacy in combination can exceed the efficacy of either drug productwhen used alone. The pharmaceutical compositions of the disclosure canbe administered alone or in combination or conjunction with interferon,ribavirin, and/or a variety of small molecules that are being developedagainst both viral targets (viral proteases, viral polymerase, and/orassembly of viral replication complexes) and host targets (hostproteases required for viral processing, host kinases required forphosphorylation of viral targets such as NS5A, and inhibitors of hostfactors required to efficiently utilize the viral internal ribosomeentry site, or IRES).

The pharmaceutical compositions disclosed herein could be used incombination or conjunction with adamantane inhibitors, neuraminidaseinhibitors, alpha interferons, non-nucleoside or nucleoside polymeraseinhibitors, NS5A inhibitors, antihistamines, protease inhibitors,helicase inhibitors, P7 inhibitors, entry inhibitors, IRES inhibitors,immune stimulators, HCV replication inhibitors, cyclophilin Ainhibitors, A3 adenosine agonists, and/or microRNA suppressors.

Cytokines that could be administered in combination or conjunction withthe pharmaceutical compositions disclosed herein include interleukin(IL)-2, IL-12, IL-23, IL-27, or IFN-γ.

New HCV drugs that are, or will be, available for potentialadministration in combination or conjunction with the pharmaceuticalcompositions disclosed herein include ACH-1625 (Achillion); Glycosylatedinterferon (Alios Biopharma); ANA598, ANA773 (Anadys Pharm); ATI-0810(Arisyn Therapeutics); AVL-181 (Avila Therapeutics); LOCTERON® (Biolex);CTS-1027 (Conatus); SD-101 (Dynavax Technologies); Clemizole (EigerBiopharmaceuticals); GS -9190 (Gilead Sciences); GI-5005 (GlobalImmuneBioPharma); Resiquimod/R-848 (Graceway Pharmaceuticals); Albinterferonalpha-2b (Human Genome Sciences); IDX-184, IDX-320, IDX-375 (Idenix);IMO-2125 (Idera Pharmaceuticals); INX-189 (Inhibitex); ITCA-638(Intarcia Therapeutics); ITMN-191/RG7227 (Intermune); ITX-5061, ITX-4520(iTherx Pharmaceuticals); MB11362 (Metabasis Therapeutics); Bavituximab(Peregrine Pharmaceuticals); PSI-7977, RG7128, PSI-938 (Pharmasset);PHX1766 (Phenomix); Nitazoxanide/ALINIA® (Romark Laboratories); SP-30(Samaritan Pharmaceuticals); SCV-07 (SciClone); SCY-635 (Scynexis);TT-033 (Tacere Therapeutics); Viramidine/taribavirin (ValeantPharmaceuticals); Telaprevir, VCH-759, VCH-916, VCH-222, VX-500, VX-813

(Vertex Pharmaceuticals); and PEG-INF Lambda (Zymogenetics).

New influenza and WNV drugs that are, or will be, available forpotential administration in combination or conjunction with thepharmaceutical compositions disclosed herein include neuraminidaseinhibitors (Peramivir, Laninamivir); triple therapy—neuraminidaseinhibitors, ribavirin, and amantadine (ADS-8902); polymerase inhibitors(Favipiravir); reverse transcriptase inhibitor (ANX-201); inhaledchitosan (ANX-211); entry/binding inhibitors (Binding Site Mimetic,Flucide); entry inhibitor, (Fludase); fusion inhibitor, (MGAWN1 forWNV); host cell inhibitors (lantibiotics); cleavage of RNA genome (RNAi,RNAse L); immune stimulators (Interferon, Alferon-LDO; Neurokinin)agonist, Homspera, Interferon Alferon N for WNV); and TG21.

Other drugs for treatment of influenza and/or hepatitis that areavailable for potential administration in combination or conjunctionwith the pharmaceutical compositions include PEGinterferon alfa-2a(Pegasys), PEGinterferon alfa-2b (Peg-Intron), ribavirin (Copegus;Rebetol), oseltamivir (Tamiflu), zanamivir (Relenza), amantadine, andrimantadine.

These agents can be incorporated as part of the same pharmaceuticalcomposition or can be administered separately from the compounds of thedisclosure, either concurrently or in accordance with another treatmentschedule.

The compounds or pharmaceutical compositions can be additive orsynergistic with other compounds or pharmaceutical compositions toenable vaccine development. By virtue of their antiviral and immuneenhancing properties, the compounds can be used to affect a prophylacticor therapeutic vaccination. The compounds need not be administeredsimultaneously or in combination with other vaccine components to beeffective. The vaccine applications of the compounds are not limited tothe treatment of viral infection but can encompass all therapeutic andprophylactic vaccine applications due to the general nature of theimmune response elicited by the compounds.

A “vaccine” is an immunogenic preparation that is used to induce animmune response in an individual. A vaccine can have more than oneconstituent that is immunogenic. A vaccine can be used for prophylacticand/or therapeutic purposes. A vaccine does not necessarily have toprevent viral infections. Without being bound by theory, the vaccines ofthe disclosure can affect an individual's immune response in a mannersuch that viral infection occurs in a lesser amount (including not atall) or such that biological or physiological effects of the viralinfection are ameliorated when the vaccine is administered as describedherein. As used herein, vaccines include preparations includingpharmaceutical compositions including the compounds, alone or incombination with an antigen, for the purpose of treating a viralinfection in a subject including a vertebrate animal.

The disclosure provides for the use of the compounds and pharmaceuticalcompositions as adjuvants. An adjuvant enhances, potentiates, and/oraccelerates the beneficial effects of another administered therapeuticagent. In particular embodiments, the term “adjuvant” refers tocompounds that modify the effect of other agents on the immune system.Adjuvants that possess this function may also be inorganic or organicchemicals, macromolecules, or entire cells of certain killed bacteria,which enhance the immune response to an antigen. They may be included ina vaccine to enhance the recipient's immune response to the suppliedantigen.

As is understood by one of ordinary skill in the art, vaccines can beagainst viruses, bacterial infections, cancers, etc. and can include oneor more of a live attenuated vaccine (LAIV), an inactivated vaccine(IIV; killed virus vaccine), a subunit (split vaccine); a sub-virionvaccine; a purified protein vaccine; or a DNA vaccine. Appropriateadjuvants include one or more of water/oil emulsions, non-ioniccopolymer adjuvants, e.g., CRL 1005 (Optivax; Vaxcel Inc., Norcross,Ga.), aluminum phosphate, aluminum hydroxide, aqueous suspensions ofaluminum and magnesium hydroxides, bacterial endotoxins,polynucleotides, polyelectrolytes, lipophilic adjuvants and syntheticmuramyl dipeptide (norMDP) analogs such asN-acetyl-nor-muranyl-L-alanyl-D-isoglutamine,N-acetyl-muranyl-(6-O-stearoyl)-L-alanyl-D-isoglutamine, orN-Glycol-muranyl-LalphaAbu-D-isoglutamine (Ciba-Geigy Ltd.).

The present disclosure further includes the use and application of thecompounds and pharmaceutical compositions in vitro in a number ofapplications including developing therapies and vaccines against viralinfections, research in modulation of the innate immune response ineukaryotic cells, etc. The compounds and pharmaceutical compositionsdisclosure can also be used in animal models. The results of such invitro and animal in vivo uses of the compounds and pharmaceuticalcompositions can, for example, inform their in vivo use in humans, orthey can be valuable independent of any human therapeutic orprophylactic use.

The Examples below are included to demonstrate particular embodiments ofthe disclosure. Those of ordinary skill in the art should recognize inlight of the present disclosure that many changes can be made to thespecific embodiments disclosed herein and still obtain a like or similarresult without departing from the spirit and scope of the disclosure.For example, the Examples below provide in vitro methods for testing thecompounds of the disclosure. Other in vitro and/or in vivo virusinfection models include flaviviruses such as DNV, bovine diarrhealvirus, WNV, and GBV-C virus, other RNA viruses such as RSV, SARS, andthe HCV replicon systems. Furthermore, any appropriate cultured cellcompetent for viral replication can be utilized in the antiviral assays.

EXPERIMENTAL EXAMPLES Example 1. General Synthesis Methods

The compounds of the disclosure may be prepared by the methods describedbelow, together with synthetic methods familiar to those of ordinaryskill in the art. The starting materials used herein are commerciallyavailable or may be prepared by routine methods known in the art (suchas those methods disclosed in standard reference books such as theCOMPENDIUM OF ORGANIC SYNTHETIC METHODS, Vol. I-VI (published byWiley-Interscience)). Preferred methods include those described below.

During any of the following synthetic sequences it may be necessaryand/or desirable to protect sensitive or reactive groups on any of themolecules concerned. This can be achieved by means of conventionalprotecting groups, such as those described in T. W. Greene, ProtectiveGroups in Organic Chemistry, John Wiley & Sons, 1981; T. W. Greene andP. G. M. Wuts, Protective Groups in Organic Chemistry, John Wiley &Sons, 1991, and T. W. Greene and P. G. M. Wuts, Protective Groups inOrganic Chemistry, John Wiley & Sons, 1999.

Compounds of the disclosure, or their pharmaceutically acceptable salts,can be prepared according to the reaction Schemes described below. Thesemethods can be modified or adapted in ways known to chemists of ordinaryskill in order to achieve synthesis of additional compounds within thescope of the present invention. Such modification was done to synthesizean exemplary compound of the invention as described in Examples 2 and 3.Unless otherwise indicated, the substituents in the Schemes are definedas above. Isolation and purification of the products is accomplished bystandard procedures, which are known to a chemist of ordinary skill.

It will be understood by one skilled in the art that the varioussymbols, superscripts and subscripts used in the schemes, methods andexamples are used for convenience of representation and/or to reflectthe order in which they are introduced in the schemes, and are notintended to necessarily correspond to the symbols, superscripts orsubscripts in the appended claims. The schemes are representative ofmethods useful in synthesizing the compounds of the present invention.They are not to constrain the scope of the disclosure in any way.

Synthesis of N-(4-hydroxy-2-methyl-1,3 -benzothiazol-5-yl)acetamide

0.6 g of commercially available 5-azido-2-methyl-1,3-benzothiazole and 5g of acetic acid were heated to 100° C. for 20 minutes. Evaporation andcolumn chromatography purification of the residue afforded 0.43 g ofN-(4-hydroxy-2-methyl-1,3-benzothiazol-5-yl)acetamide.

Synthesis of 5-amino-2-methyl-1,3-benzothiazol-4-ol

0.4 g of the acetamide was treated with 2 mL concentrated HCl.Evaporation provided 0.38 g of 5-amino-2-methyl-1,3-benzothiazol-4-ol asthe di-HCl salt.

Synthesis of 7-methyl[1,3]thiazolo[5,4-g][1,3]benzoxazol-2-amine

To a solution of 5 mL 3:4 methanol:water solution cooled to 0° C. wasadded 0.08 mL bromine followed by 0.12 g of KCN in portions. When thebromine color was gone the cyanogen bromide solution was added to 0.38 gof the amine dihydrochloride in 20 mL water and 0.252 g sodiumbicarbonate and the reaction was left overnight. The reaction wasfiltered and the filtrate was treated with sodium bicarbonate andconcentrated under vacuum. The residue was dissolved in ethanol and thesolution was filtered. The filtrate was concentrated to a residue whichwas purified by chromatography to afford 0.14 of7-methyl[1,3]thiazolo[5,4-g][1,3]benzoxazol-2-amine.

Example 2. Synthesis ofN-(7-methyl[1,3]thiazolo[5,4-g][1,3]benzoxazol-2-yl)thiophene-2-carboxamide

Acid chloride coupling was performed:

To a suspension of 0.15 g7-methyl[1,3]thiazolo[5,4-g][1,3]benzoxazol-2-amine in 2.5 mL drypyridine was added 0.078 mL thiophene-2-carbonyl chloride. The reactionwas stirred for 5 h at 80° C. then cooled to room temperature. 4 mL ofwater was added and the precipitate was filtered off, washed with waterand dried to afford 0.154 g ofN-(7-methyl[1,3]thiazolo[5,4-g][1,3]benzoxazol-2-yl)thiophene-2-carboxamide.

Example 3. Synthesis ofN□[6□(pyrrolidine□1□sulfonyl)□1,3□benzothiazol□2□yl]naphthalene□2□carboxamide

The intermediate 6-(pyrrolidin-1-ylsulfonyl)-1,3-benzothiazol-2-amine,which was used in the synthesis ofN□[6□(pyrrolidine□1□sulfonyl)□1,3□benzothiazol□2□yl]naphthalene□2□carboxamide,was synthesized as described below:

A mixture of commercially available 4-(pyrrolidin-1-ylsulfonyl)aniline(1.0 g) and ammonium thiocyanate (1.01 g) was suspended in 25 mL aceticacid and heated to 90° C. The mixture was cooled to 15° C. and liquidbromine (0.22 mL) was added dropwise. The reaction was stirred at roomtemperature overnight then filtered. The filtrate was concentrated undervacuum and the residue was added to a solution of aqueous sodiumbicarbonate and stirred for 1 hour. The precipitate was filtered off,washed with water and ether and dried to afford 0.7 g of6-(pyrrolidin-1-ylsulfonyl)-1,3-benzothiazol-2-amine.

0.1 g of 6-(pyrrolidin-1-ylsulfonyl)-1,3-benzothiazol-2-amine wasdissolved in 2 mL dry pyridine and 2-naphthoyl chloride (0.067 g) wasadded and the mixture was stirred at 80° C. for 5 h. After cooling toroom temperature the mixture was added to 0.7 mL water and theprecipitate was filtered off, washed with water and ether and dried toafford 0.091 g ofN□[6□(pyrrolidine□1□sulfonyl)□1,3□benzothiazol□2□yl]naphthalene□2□carboxamide:

Example 4. Antiviral Activity and Pharmacological Properties usingStructure-Activity Relationship (SAR) Studies

This Example describes optimization of compounds for antiviral action.First, a small analog derivative set is used to define a structuralclass. The active analogs that are identified in this first stage arethen used to define a subset of structural classes of interest forfurther optimization (Stage 2).

Stage 2 focuses on creating structural diversity and evaluating corevariants for derivative expansion. Structural derivatives are tested forbiological activity including the IRF-3 translocation assay, antiviralactivity, and cytotoxicity in one or more cell lines or peripheral bloodmononuclear cells. Optimized molecules that show improved efficacy andlow cytotoxicity are further characterized by additional measures of invitro toxicology and absorption, distribution, metabolism, andelimination (ADME). Their mechanism of action and breadth of antiviralactivity are also studied.

To design analog structures, the drug-like properties, metaboliclability, and toxic potential of the lead compounds are analyzed.Drug-like properties, as measured by Lipinski's Rules, and relatedphysiochemical properties are primary indicators of bioavailability.Structural features that suggest metabolic and toxicological liabilitiesmay indicate limited stability, reduced half-life, reactiveintermediates, or idiosyncratic toxicity and will therefore be removed.

Compounds are tested for potent in vitro antiviral activity againstviruses including HCV 2A, RSV, DNV type 2, and influenza A virusstrains. Viral protein and RNA levels are assessed following drugtreatment using the assays described herein. Analog design is aimed toidentify lead compounds with picomolar to nanomolar potency, which isadequate to support preclinical development Lead compounds arecharacterized for their in vitro toxicological and ADME properties andfor further mechanistic study.

In vitro pharmacology studies are performed to measure performance ofthe most promising analogs in one or more assays of intestinalpermeability, metabolic stability and toxicity. Key in vitrocharacterization studies can include plasma protein binding; serum,plasma, and whole-blood stability in human and model organisms;intestinal permeability; intrinsic clearance; human Ether-à-go-go (hERG)channel inhibition; and genotoxicity.

For each analog, an HPLC- and/or HPLC-mass spectrometry-based analyticalmethod is used to evaluate drug and metabolite concentrations in varioustest systems. Although the specific analytical method is optimized foreach molecule, reverse-phase chromatography can be used alone or incombination with quadrupole mass spectrometry to characterize theidentity and purity of several of the lead molecules. Initially, drugstability over time in increasing concentrations of serum, plasma, andwhole blood from mammalian species (such as mouse, cynomolgus macaque,and human) is evaluated by HPLC, and a half-life is determined.

Prominent metabolites were characterized by mass spectrometry. Humanplasma protein binding were evaluated by partition analysis usingequilibrium dialysis. For intestinal permeability modeling,apical-to-basolateral flux is assessed in the human epithelial cell lineTC7. Hepatic clearance is estimated for a subset of the most promisinganalogs by measuring the rate of disappearance of the parent compoundduring incubation in human liver microsomes. As above, specificmetabolites can be isolated and characterized.

In vitro toxicology studies are performed to evaluate the potentialcardiac and genetic toxicity of lead analogs. Automated patch-clamp isused to assess the impact of each compound on hERG channel currents in arecombinant Chinese hamster ovary (CHO) cell line transgenicallyexpressing the human Kv11.1 gene. Concentrations up to the lesser of 30times the maximum serum concentration or the limit of solubility of eachcompound are evaluated in order to determine an IC50 for the molecule onthe hERG channel. A subset of compounds is evaluated over a range ofconcentrations for their ability to induce mutation reversion inSalmonella typhimurium strains TA98 and TA100 or to promote micronucleusformation in CHO cells in culture.

Example 5. Biological Activity

This Example describes methods used to identify compounds that activateinnate immune responses, including activation of the RIG-I pathway.Other compounds as described herein likewise can be evaluated by themethods described in this example, and other cell types can also beused.

Cultured Huh 7 cells that were stably transfected with a luciferasereporter gene coupled with a RIG-I signaling pathway responsive promoter(IFβ, ISG56, or ISG54 promoter) were seeded and allowed to growovernight. The compound 1 was then added and cells were grown in thepresence of compound 1 for 18-20 hours. Steady-Glo luciferase substrate(Promega) was added and luminescence was read on a luminometer(Berthold).

FIG. 1A shows that compound 1 of Table 1 as described herein wasvalidated by demonstrating dose-dependent induction of the luciferasereporter gene coupled to the promoters for IFNβ (“IFNβ-LUC,” left),ISG56 (“ISG56-LUC,” center), and ISG54 (“ISG54-LUC,” right).Additionally, compound 1 did not induce a nonspecific promoter(β-actin-LUC, FIG. 1B).

An immunofluorescent cytochemistry assay was used to determine IRF-3activation and translocation to the nucleus. Cultured human HeLa cellswere treated with increasing amounts of compound or equivalent amountsof DMSO diluted in media for 20 hours. Positive control wells wereinfected with 100 HA/mL Sendai virus for an equivalent time period.IRF-3 was detected using polyclonal rabbit serum specific to IRF-3 and asecondary antibody conjugated to DYLIGHT® (Pierce Biotechnology, Inc.,Rockford, Ill.) 488.

An immunofluorescent cytochemistry assay was used to determine NFκBactivation. The innate immune response is also dependent on activationof the NFκB transcription factor. Cultured human HeLa cells were treatedwith increasing amounts of compound or equivalent amounts of DMSOdiluted in media for 20 hours. Positive control wells were infected with100 HA/mL Sendai virus for an equivalent time period. NFκB was detectedusing monoclonal mouse antibody specific to the p65 subunit of NFκB anda secondary antibody conjugated to DyLight 488.

Quantification of the IRF-3 and NFκB immunofluorescent assays describedherein was done as follows: 96-well plates containing cultured humancells treated with compound and stained for either IRF-3 or NFκB werescanned and quantified using the ARRAYSCAN® instrument and software(Cellomics). Activation of transcription factor was evidenced byincreased nuclear intensity normalized for cytoplasmic intensity, ornuclear-cytoplasmic difference. Compound 1 shows a dose dependentincrease in nuclear-cytoplasmic difference for IRF-3 (FIG. 1C) and forNFκB (FIG. 1D).

Assays for innate immune gene expression were performed in cell typesincluding HeLa cells, PH5CH8 cells, and HUVEC primary cells. Geneexpression can be similarly assayed in cell types that include: primaryblood mononuclear cells, human macrophages, THP-1 cells, Huh 7 cells,A549 cells, MRCS cells, rat splenocytes, rat thymocytes, mousemacrophages, mouse splenocytes, and mouse thymocytes. Expression ofother genes of interest can be assayed as described herein.

Cultured HeLa cells were treated with 20 μM, 10 μM, 5 μM of compound ora DMSO control and incubated for up to 24 hours. Cultured PH5CH8 cellswere treated with 10 μM, 5 μM, 1 μM, or a DMSO control and incubated forup to 24 hours. Primary HUVEC cells were thawed and seeded in 6-wellplates at 2.4×104 cells per well and allowed to grow to 80% confluence,typically 5 days in culture with fresh media replaced every 48 hours.Compound was added at 10 μM, 1 μM or a DMSO control and incubated for upto 24 hours. Gene expression was assayed as described below.

Cells were harvested and RNA was isolated using the QIAshredder columnsand RNeasy Mini Kit (Qiagen) according to manufacturer instructions.Reverse transcription was performed and the cDNA template was used forquantitative real-time PCR. PCR reactions were performed usingcommercially available, validated TaqMan gene expression assays (AppliedBiosystems/Life Technologies) according to manufacturer instructions.Gene expression levels were measured using a relative expressionanalysis (ΔΔCt).

FIGS. 2A-2C show induction of gene expression by compounds 1 and 2 ofTable 1. FIG. 2A shows gene expression levels of IFIT2 (left) and OAS1(right) in HeLa cells over time from 4-24 hours post treatment with 10μM compound 1 (grey; OAS1 only) or 10 μM compound 2 (black; IFIT2 andOAS1 both shown). FIG. 2B shows gene expression levels of IFIT2 inPH5CH8 cells (solid color bars) and HeLa cells (black checked bars)treated with 10 μM compound 1 (CPD 1) or compound 2 (CPD 2). FIG. 2Cshows gene expression levels of IFIT2 (left), OAS1 (center), and M×A(right) in primary HUVEC cells that were treated with compound 1 (CPD 1)or 1 μM compound 2 (CPD 2).

FIGS. 3A-3B show induction of gene expression by compound 3 and compound7 of Table 1. FIG. 3A shows IFIT2 gene expression was induced by 5 μMcompound 3 or compound 7. FIG. 3B shows compound 3 induced innate immunegene expression in mouse macrophage cells.

Example 6. Ex Vivo Immune Stimulatory Activity of Compound 1

The activity of compound 1 of Table 1 in primary immune cells wasassayed to determine whether compound 1 stimulates immune responses.Cultured human primary dendritic cells were treated with 0, 1, or 10 μMof compound 1 for 24 hours. Supernatant from treated wells was isolatedand tested for levels of cytokine protein. Cytokines were detected usingspecific antibodies conjugated to magnetic beads and a secondaryantibody that reacts with Streptavidin/Phycoerythrin to produce afluorescent signal. The bound beads were detected and quantified usingthe MAGPIX® (Luminex Corp.) instrument, although similar techniques asare known in the art may be used to measure fluorescent proteinproduction, such as for example an ELISA.

FIG. 4 shows induction of the chemokines IL-8, MCP-1, MIP-1α, and MIP-1βby dendritic cells treated with compound 1 of Table 1 (concentrationsshown in μM). LPS is shown as a positive control inducer of chemokineexpression.

Other cells from which cytokine secretion can be measured include, forexample human peripheral blood mononuclear cells, human macrophages,mouse macrophages, mouse splenocytes, rat thymocytes, and ratsplenocytes.

Example 7. In Vitro Antiviral Activity

To further characterize the breadth of antiviral activity of optimizedmolecules, cell culture infection models are used to analyze differentviruses, including different strains of influenza virus, HCV, DNV, RSV,and WNV, an emerging public health concern. The studies include treatingcells with compound 2-24 hours prior to infection or treating cells upto 8 hours after infection. Virus production and cellular ISG expressionare assessed over a time course to analyze antiviral effects ofrepresentative compounds from lead structural classes. Known antiviraltreatments including IFNβ are used as a positive control.

Virus production is measured by focus-forming or plaque assay. Inparallel experiments, viral RNA and cellular ISG expression are measuredby qPCR and immunoblot analyses. These experiments are designed tovalidate compound signaling actions during virus infection, and assesscompound actions to direct innate immune antiviral programs againstvarious strains of viruses and in the setting of virus countermeasures.Detailed dose-response analyses of each compound are conducted in eachvirus infection system to determine the effective dose that suppressesvirus production by 50% (IC50) and 90% (IC90) as compared with controlcells for both the pre-treatment and post-treatment infection models.

Compounds of the current invention are tested using in vitro modelsagainst viruses including: Hepatitis B Virus (HBV), HCV H77 (genotypela), HVV JFH1 (genotype 2a), Influenza A/PR/8/34 (H1N1 mouse-adaptedvirus), Influenza A/WSN/33 (H1N1 mouse-adapted neurovirulent virus),Influenza A/TX/36/91 (H1N1 circulating virus), Influenza A/Udorn/72(H3N2), WNV TX02 (lineage 1), WNV MAD78 (lineage 2), RSV, humancoronavirus OC43 (SARS-like pathogen), and DNV type 2.

Antiviral activity of exemplary compounds of the disclosure isdemonstrated in Examples 8-11 below.

Example 8. In Vitro Activity Against Respiratory Syncytial Virus

HeLa cells were seeded the previous day in 6-well plates at 4×10⁵ cellsper well. The next day, the media was replaced with RSV in media withoutFBS at an MOI of 0.1. Virus binding occurred at 37° C. for 2 hours.After 2 hours the cells were washed with warm complete media andreplaced with media containing drug at varying concentrations of 10 μM,5 μM, 1 μM, or a DMSO control. Cells were placed in a 37° C. incubatorfor 48 hours.

For virus detection and titration, HeLa cells were seeded in 96-wellplates at 8×10³ cells per well 24 hrs prior to collecting virussupernatant. After the 48 hour incubation period, the virus supernatantfrom the infected plate was harvested and used to infect these cells ata 1/10 final dilution. Cells were placed in a 37° C. incubator for 24hours.

24 hours after infection, cells were washed twice with PBS and fixedwith methanol/acetone solution. After fixing the cells were washed twicewith PBS and replaced with blocking buffer (10% horse serum, 1g/mL BSAand 0.1% Triton-100× in PBS) for 1 hour. The blocking buffer wasreplaced with binding buffer containing a 1/2000 dilution of primaryantibody for 2 hours at room temperature. The primary antibody was amouse monoclonal antibody against RSV. The cells were washed twice withPBS and replaced with binding buffer containing 1/3000 dilution of theAlexa Fluor-488 goat anti-mouse secondary antibody and a Hoechst nuclearstain for 1 hour at room temperature. The cells were washed twice withPBS and PBS is added to all wells. The 96-well plate was sealed andfluorescence activity associated with virus infectivity was determinedby immunofluorescent assay using the Array Scan instrument(Thermo-Fischer).

Treatment with compounds can be done prior to infection. In variationsof this method, the compounds are added at varying time points prior toinfection with virus. Virus detection and titration is conducted asdescribed.

FIG. 5 shows results of experiments performed using the protocol ofExample 8, demonstrating the antiviral activity of select compounds thatdemonstrated antiviral activity against RSV. +++=greater than 70%inhibition of infection, ++=greater than 50% inhibition, +=greater than30% inhibition, −=less than 30% inhibition.

Example 9. In Vitro Activity Against Influenza Virus

Influenza A/Udorn/72 infection of H292 cells. 2×10⁶ H292 cells inRPMI1640+10%FCS were treated with 2 μM compound 2 in a finalconcentration of 0.5% DMSO for 6 hours. Compound-containing media wasaspirated and replaced with 1× MEM containing A/Udorn/72 at an MOI of0.1 and placed at 37° C. in a CO² incubator. Two hours post infection,virus-containing media was aspirated and replaced with 1× MEM containinglug/mL TPCK-treated Trypsin, 2 μM compound 2, 0.5% DMSO. Cells wereplaced in 37° C. CO₂ incubator for 18 hours. After 20 hourspost-infection, virus supernatants were collected and titered on MDCKcells.

Influenza A/Udorn/72 infection of HEK293 cells. 5×105 HEK293 cells wereinfected with A/Udorn/72 at an MOI of 0.2 in 1× MEM. After 2 hourspost-infection, virus-containing media was aspirated and replaced with1× MEM containing 1μg/mL TPCK-treated Trypsin, 10 μM compound 2, 0.5%DMSO. Cells were returned to 37° C., CO2 incubator for 18 hours. After20 hours post-infection, virus supernatants were collected and titred onMDCK cells.

Titer in MDCK cells. 10pL of infected supernatant was added to 2×106MDCK cells in the presence of 2 μg/mL TPCK-trypsin and placed in a 37°C. CO2 incubator. After 8 hours, supernatant was removed and cells werefixed and stained with FITC-conjugated antibody specific for InfluenzaNP protein. Number of foci was quantitated using the ArrayScaninstrument and software (Cellomics).

The protocol of Example 9 can be performed as described in order toevaluate the antiviral activity of example compounds. FIG. 6 showsantiviral activity of example compounds against Influenza A virusUdorn/72. Treatment of HEK293 cells with increasing concentrations ofcompound 3, compound 7, compound 9, and compound 10 of Table 1 resultedin dose-dependent inhibition of virus infection (shown as % untreatednegative control). Calculated IC50 values are shown. Table 2 showscalculated IC50 values of selected compounds from Table 1.

TABLE 2 IC50 against flu Compound IC50 (μM) 2 2.04 3 0.61 4 1.29 5 >10 61.49 7 >5 9 1.15 10 3.18 11 5.16 12 2 13 2 15 6.8

Example 10, In Vitro Activity Against Dengue Virus

Cultured human Huh 7 cells were seeded in 6-well tissue-culture platesat a density of 4×10⁵ cells per well and grown for 24 hours. Cells wereinfected with DNV type 2 strain at multiplicity of infection (MOI) of0.1 for 2 hours and then removed. Compound dilutions were prepared in0.5% DMSO and used to treat cells at final concentrations of compoundranging 0.001 to 10 μM per well. Vehicle control wells treated with 0.5%DMSO were used to compare to drug treated cells. Replication was allowedto proceed for 48 hours. Virus supernatants were harvested and used toinfect new monolayer of permissive cells, such as Vero cells that wereseeded in 96-well plates at 8×10³ cells per well 24 hrs prior tocollecting virus supernatant.

The newly infected cells were incubated for 24 hours and used to measurethe level of infectious virus in the original supernatants byimmunofluorescent staining of viral protein. The cells were fixed withice-cold 1:1 methanol and acetone solution and stained for DNV fusionprotein. Primary mouse monoclonal antibody against DNV fusion protein(Millipore) was used at 1:2000 dilution. Secondary goat anti-mouseantibody conjugated to Alexa Fluor 488 dye (Invitrogen) and Hoescht Dye(nuclear staining) are used at 1:3000 to detect DNV protein and cellnuclei. Following secondary antibody incubation, the monolayers werewashed and left in 100 μL PBS for imaging and quantitation using aCellomics ArrayScan HCS instrument.

FIG. 7 shows the antiviral activity of compound 5 and compound 20 ofTable 1 against Dengue virus (DNV) type 2. Treatment with increasingamounts of compound showed dose-dependent decrease in infection byvirus.

FIG. 8 shows antiviral activity of exemplary compounds against Denguevirus type 2. Treatment of Huh 7 cells with increasing concentrations ofcompound 8, compound 3, compound 5, compound 6, compound 7, compound 9,and compound 10 of Table 1 resulted in dose-dependent inhibition ofvirus infection (shown as % untreated negative control). Calculated IC50values are shown.

Table 3 shows calculated IC50 values of selected compounds againstDengue virus type 2 (DV2) and/or Dengue virus type 4 (DV4).

TABLE 3 IC50 against DNV Compound IC50 against DV2 (μM) IC50 against DV4(μM) 2 6.18 3 1.87 0.78 4 3.65 5 0.47 4.9 6 2.03 0.02 7 0.74 1.87 8 1.239 1.78 10 1.78 6.48 11 0.53 21 0.10 0.27 12 0.14 0.15 14 0.15 0.03 150.39 >5 16 >5 17 0.50 4.7

Example 11. In Vitro Activity Against Human Coronavirus

MRCS cells were seeded the previous day in 6-well plates and grown for24 hours. Cells were infected with human coronavirus OC43 (HCoV-OC43)for 2 hours and then removed. Compound dilutions were prepared in 0.5%DMSO and used to treat cells at final concentrations of compound ranging0.001 to 10 μM per well. Vehicle control wells treated with 0.5% DMSOwere used to compare to drug treated cells. Replication was allowed toproceed for 5 days. Virus supernatants were harvested and used to infectnew monolayer of permissive cells, such as Huh 7 cells that were seededin 96-well plates 24 hours prior to collecting virus supernatant, i.e.,4 days post infection.

The newly infected cells were incubated for 48 hours and used to measurethe level of infectious virus in the original supernatants byimmunofluorescent staining of viral protein. The cells are fixed withice-cold 1:1 methanol and acetone solution and stained for HCoV-OC43nucleoprotein. Primary mouse monoclonal antibody against HCoV-OC43nucleoprotein (Millipore) is used at 1:1000 dilution. Secondary goatanti-mouse antibody conjugated to Alexa Fluor 488 dye (Invitrogen) andHoescht Dye (nuclear staining) were used at 1:3000 to detect OC43protein and cell nuclei. Following secondary antibody incubation, themonolayers were washed and left in 100 μL PBS for imaging andquantitation using a Cellomics ArrayScan HCS instrument.

FIG. 9 shows antiviral activity of exemplary compounds against humancoronavirus OC43. Treatment with increasing concentrations of compound3, compound 5, compound 6, and compound 7 of Table 1 resulted indose-dependent inhibition of virus infection (shown as % untreatednegative control). Calculated IC50 values are shown.

Table 4 shows calculated IC50 values of selected compounds against humancoronavirus OC43.

TABLE 4 IC50 against OC43 Compound IC50 (μM) 2 9.22 3 0.54 4 0.09 5 0.046 0.04 7 0.02

Example 12. In Vivo Pharmacokinetic and Toxicological Properties ofCompounds

The in vivo pharmacokinetic (PK) profile and tolerability/toxicity ofthe compounds described herein are evaluated in order to conduct furthercharacterization of their antiviral activity in vivo.

A reverse-phase, HPLC-MS/MS detection method is used for measuring theconcentration of each compound in mouse plasma. Prior to PK profiling,an initial oral and intravenous formulation for each compound isdeveloped using a limited formulation component screen that is largelyfocused on maximizing aqueous solubility and stability over a smallnumber of storage conditions. Any of the analytical methods as are knownin the art can be used to measure formulation performance. A formulationis developed for each compound following a three tiered strategy:

-   -   Tier 1: pH (pH 3 to 9), buffer, and osmolality adjustment    -   Tier 2: addition of ethanol (<10%), propylene glycol (<40%), or        polyethylene glycol (PEG) 300 or 400 (<60%) co-solvents to        enhance solubility    -   Tier 3: addition of N-N-dimethylacetamide (DMA, <30%),        N-methyl-2-pyrrolidone (NMP, <20%), and/or dimethyl sulfoxide        (DMSO, <20%) co-solvents or the cyclodextrins (<40%) as needed        to further improve solubility.

Example 13. In Vivo Antiviral Activity of Compounds

For compounds that demonstrate adequate performance in in vitroantiviral, mechanistic, ADME, and toxicology studies, a preliminarymouse PK study is performed. Each compound is administered as a singledose to animals by oral gavage (<10 ml/kg) or i.v. bolus injection (<5ml/kg) after an overnight fast. Multiple animals are dosed for eachdosing group such that 3 animals can be sampled at each time point.Blood samples are collected by retro-orbital sinus prior to dosing andat 5, 15, and 30 minutes, and 1, 2, 4, 8, and 24 hours post-dosing. Drugconcentrations are measured according to the previously developedbioanalytical method. PK parameters are evaluated using the WinNonlinsoftware.

Based upon performance in exploratory PK studies, compounds are furtherevaluated for preliminary tolerability and toxicity in mice prior totheir characterization in antiviral models. Tolerability studies areperformed in two stages: an initial dose escalation stage (up to 5doses, each separated by a 5-day washout period) to determine themaximum tolerable dose (MTD, Phase 1), followed by seven dailyadministrations of the MTD to evaluate acute toxicity (Stage 2). Alldoses are administered by oral gavage. In an example experiment, fiveanimals of each sex are placed on-study in stage 1 and 15 animals persex per dosing group in Stage 2. Study endpoints include a determinationof the MTD, physical examination, clinical observations, hematology,serum chemistry and animal bodyweights. Gross pathology is performed onall animals whether found dead, euthanized in extremis, or at theintended conclusion of the experiment. The toxicology studies areprimarily exploratory in nature and intended to identify earlytoxicological endpoints, and drive selection of lead candidates forantiviral animal models.

Example methods to complete the PK and tolerability studies describedabove are shown in Table 5. These methods may be modified and/or adaptedsuch as a different route of administration, in order to more accuratelymeasure the pharmacological properties of a compound.

TABLE 5 Experimental Route of Study design administration Outcomes MousePK Single dose IV and Oral Oral bioavailability, pharmacokineticC_(max), t_(1/2), Cl, study V_(d), AUC_(0-24, 0-∞) Mouse Phase 1: OralMTD, acute toxicity, tolerability ascending dose hematology, serumtolerability chemistry, gross and MTD pathology determination; Phase 2:placebo controlled 7-day toxicity at MTD

FIG. 10 shows results from exploratory PK studies. Administration ofcompound 3 of Table 1 via oral (PO) or intravenous (IV) route resultedin detectable levels of compound in serum samples obtained up to 250minutes post treatment (FIG. 10A). At 4 hours post treatment of compound3 and compound 7 of Table 1, there was detectable compound in lungtissue (FIG. 10B).

Example compounds of the disclosure that demonstrate desirable PKproperties, tolerability, antiviral potency, and/or innate immuneactivating activity, are selected for further evaluation in preclinicalmouse models of infection.

Incorporated in the design of these experiments is the determination ofan effective dose for 50% and 90% suppression of serum viral load (EC50and EC90) by each compound after a standard challenge of virus; forexample, 100 pfu of WNV-TX or 1,000 pfu of influenza virus. Virusquantification in serum and/or target tissues are determined byestablished assay methods including: plaque assay, TCID50 assay, focusforming assay, viral protein quantification such as through HA assay orBCA assay, viral RNA quantification such as through qPCR, and/or antigenquantification such as through ELISA.

The compound actions are tested in virus challenge studies at a minimumof 2 dose levels including the determined EC50 and EC90 to evaluatetheir ability to limit viral pathogenesis, virus replication, and virusspread. Mice are monitored for morbidity and mortality over a range ofchallenge doses (for example, 10 to 1,000 pfu of virus) either alone orin combination with compound treatment beginning 12 hours before or 24hours after infection and continuing daily subject to the determinedplasma half-life of the drug. Compound dose-response analysis andinfection time course studies are also conducted to evaluate compoundefficacy to: 1) limit serum viral load, 2) limit virus replication andspread in target organs, and 3) protect against viral pathogenesis.

Studies to define effective dosage of drug in vivo and established mousevirus infection models are described in Table 6, although this list isnot intended to be complete and the compounds can be tested in any mousemodel for potency against any virus infection.

TABLE 6 Experimental Study design Analysis Outcomes Compound dose Drugmeasured in Drug concentration Define in vivo determination blood at ≥3dose in blood; HPLC compound exposure levels; 2, 8, 24 reverse phasehours post treatment Effective Viral burden analysis Viral burden Definein vivo compound dose at ≥3 dose levels analysis in serum EC₅₀ and EC₉₀determination and/or target tissues Viral pathogenesis Treatment at Timeto moribund state, Define compound study 1: defined doses clinicalscoring for action toward EC₅₀ and EC₉₀ of EC₅₀ and EC₉₀ pathologicsigns of limiting viral Treatment infection pathogenesis Viralpathogenesis Treatment at Viral burden Define compound study 2: defineddoses analysis in serum action toward EC₅₀ and EC₉₀ of EC₅₀ and EC₉₀ andvarious limiting virus treatment and target organs replication and timecourse spread analysis Mouse WNV Intracranial injection Time to moribundstate, Define compound neuroinvasion of WNV-TX; drug clinical scoringfor action toward Model treatment at 2 pathologic signs of limitingviral doses w/placebo infection pathogenesis in the CNS Mouse InfluenzaIntranasal or tracheal Mortality, viral Define compound Modelinstillation of titer in serum/target action toward A/PR/8/34, organs,body temp., limiting viral A/WSN/33 or clinical observations,pathogenesis, A/Udorn/72; drug bodyweight, cytokine virus replication,treatment at 2 levels, gene expression, and spread doses w/placebomarkers of inflammation Mouse RSV Intranasal or tracheal Mortality,viral Define compound Model instillation of RSV titer in serum/targetaction toward A2 Long strain; drug organs, body temp., limiting viraltreatment at 2 doses clinical observations, pathogenesis, w/placebocontrol bodyweight, cytokine virus replication, levels, gene expression,and spread markers of inflammation Mouse DNV IP injection of DV-2;Mortality, viral Define compound Model drug treatment at 2 titer inserum/target action toward doses w/placebo organs, body temp., limitingviral clinical observations, pathogenesis, bodyweight, cytokine virusreplication, levels, gene expression, and spread markers of inflammationMouse MHV-1 Intranasal instillation Mortality, viral Define compoundModel of MHV-1; drug treatment titer in serum/target action toward at 2doses w/placebo organs, body temp., limiting viral clinicalobservations, pathogenesis, bodyweight, cytokine virus replication,levels, gene expression, and spread markers of inflammation

Mouse WNV model. Efficacy of compounds against WNV can be assayed aftersubcutaneous or intracranial (neuroinvasion) infection of virus.Compounds are administered daily by oral gavage or IP administrationover the entire course of infection at 2 dose levels plus a placebocontrol group. Animals are evaluated for study endpoints including dailyclinical observations, mortality, body weight, and body temperature.Virus titer is measured in serum, lymph nodes, spleen, and/or brain.Gene and cytokine expression at various time points during infection incompound-treated versus control animals can be assayed.

Mouse influenza model. Virus infection is done by non-surgicalintranasal or tracheal instillation of influenza virus strains A/WSN/33and A/Udorn/72. These influenza virus strains are two different subtypes(H1N1 and H3N2) and exhibit varying pathogenic properties and clinicalpresentations in C57B1/6 mice. Compounds are administered daily by oralgavage or IP administration over the entire course of infection (>2weeks) at 2 dose levels plus a placebo control group. Animals areevaluated for study endpoints including daily clinical observations,mortality, body weight, and body temperature. Virus titer is measured inserum, heart, lung, kidney, liver, and/or brain. Gene and cytokineexpression at various time points during infection in compound-treatedversus control animals can be assayed.

Mouse RSV model. Virus infection is done by non-surgical intranasal ortracheal instillation of RSV A2 long strain at a dose that does notcause cytopathic effects. Compounds are administered daily by oralgavage or IP administration for ≥3 weeks, at 2 dose levels or a placebocontrol. Animals are evaluated for study endpoints including dailyclinical observations, mortality, body weight, and body temperature.Virus titer is measured in serum, blood, and/or lung. Gene and cytokineexpression, and increased immune cell population counts can be assayed.

Mouse DNV model. Virus infection is done by intraperitoneal injection ofDNV type 2 strain. Compounds are administered daily by oral gavage or IPadministration over the entire course of infection at 2 dose levels plusa placebo control group. Animals are evaluated for study endpointsincluding daily clinical observations, mortality, body weight, and bodytemperature. Virus titer is measured in serum, blood, heart, lung,kidney, liver, and/or brain. Gene and cytokine expression at varioustime points during infection in compound-treated versus control animalscan be assayed.

Mouse hepatitis virus type 1 (MHV-1) coronavirus model. Virus infectionis done by non-surgical intranasal instillation of MHV-1. Compounds areadministered daily by oral gavage or IP administration over the entirecourse of infection (≥1 week) at 2 dose levels plus a placebo controlgroup. Animals are evaluated for study endpoints including dailyclinical observations, mortality, body weight, and body temperature.Virus titer is measured in serum, heart, lung, kidney, liver, and/orbrain. Gene and cytokine expression at various time points duringinfection in compound-treated versus control animals can be assayed.

FIG. 11 shows a study performed using the mouse hepatitis virus type 1(MHV-1) coronavirus model. Treatment with compound 3 of Table 1 resultedin decreased pathological symptoms including weight loss (A) andincreased survival (B) after lethal challenge with MHV-1. (C) Virus wasdecreased in the lung of animals treated with compound 3.

The antiviral activity of example compounds are described in Table 7.

TABLE 7 DNV2 DNV4 FLU RSV HCOV Compound EC50 EC50 EC50 EC50 EC50 ID (uM)(uM) (uM) (uM) (uM) 1 >10 2.1 >10 >10 NA 2 6.2 >5 2 >10 9.2  3 1.9 1.22.6 2.3 0.54 4 3.6 NA 1.3 2.8 NA 5 0.5 5 >10 NA 0.04 6 2.5 2.8 1.1 1.70.04 7 0.71 3.4 NA >3 0.02 8 1.2 NA NA NA NA 9 1.8 0.9 1.1 1.8 NA 10 1.45.6 3.2 2.9 NA 11 0.5 5.2 NA NA NA 12 0.2 2.8 6.8 7.25 NA 13 2.0 5.5 5.314.00 NA 15 0.4 4.6 6.8 >3 NA 16 NA NA NA NA NA 17 0.5 4.7 4.1 NA NA 190.5 0.6 NA 1.2 NA

Example 14. In Vivo Adjuvant Activity

To characterize the breadth of adjuvant activity of compounds of thedisclosure, animal models of vaccination and vaccination plus protectionare used. The studies include priming animals including rats and micewith compound alone or in combination with an antigen and then assessingthe adjuvant effect.

Adjuvant effect is measured by assays for modified, enhanced immunehumoral and cellular responses. Humoral responses are assessed over timeat discrete times post vaccination and/or boosting by collecting bloodfor sera and determining relative concentrations of antibody classes(IgM, IgG, IgA or IgE) and/or isotypes including IgG1, IgG2a, IgG2b,IgG2c, IgG3 for IgG antibodies. Moreover, affinity and avidity of thegenerated antibodies is also determined. In instances in which thevaccine preparation includes a combination of compound and antigen, theneutralizing activity of the generated antibodies is also determined.

Cellular mediated immune responses induced by the compounds are measuredby established methods in the field including ex vivo stimulation ofperipheral blood mononuclear cells, lymph nodes, splenocytes or othersecondary lymphoid organs with the antigen and measurement of cytokineor chemokine production in the supernatant at several times thereafter.Cytokines measured include Th1 type of cytokines including IFN gamma andTNF alpha, Th2 type cytokines including IL-4, IL-10, IL-5 and IL-13 andTh17 cytokines including IL-17, IL-21 and IL-23. Chemokines elicited bythe compounds are also measured including RANTES, IP-10, MIP1a, MIP1b,and IL-8. T cell antigen specific production of cytokines can also bemeasured by intracellular cytokine staining with fluorescently labeledspecific antibodies and flow cytometry or by ELISPOT. Both CD4+ ad CD8+T cell populations are studied.

Measurement of adjuvant activity at the cellular level is alsodetermined by immunophenotyping of surface markers of activation by flowcytometry. CD8 T cell antigen-specific responses are also evaluated byintracellular cytokine staining of perforin, cell surface markerexpression or proliferation assays including thymidine incorporation.

These experiments are designed to validate compound adjuvant activity indifferent combinations of prime-boost schemes and assess howcompound-induced effects on innate immune antiviral programs shape theadaptive immune responses mounted to the antigen in the vaccinepreparations.

Detailed immune response analyses of each compound as described aboveare conducted with each selected antigen to determine the immunecorrelates for that particular antigen(s) and compound formulation.These results guide the protection studies in which animals vaccinatedand boosted with combinations of select optimized compounds and desiredantigen(s) formulations from select infectious agents are laterchallenged with doses of infectious agent that are known to result indisease or death of the animal. Protection afforded by vaccination istypically measured by monitoring of clinical symptoms and survival.

Example 15. Anti-Viral Activity of Compound 12 of Table 1 Against EbolaVirus

The in vitro efficacy of compound 12 of Table 1 was tested against Ebolavirus (EBOV). As shown in FIG. 12, compound 12 showed greater than a 2log reduction in EBOC titer in vitro. Control titer (pfu/mL) was over 5,whereas the test titer using compound 12 was less than 3.5.

Example 16. Anti-Viral Effect of Compound 8 of Table 1

FIG. 13 shows the dose response activity of compound 8 of Table 1against DENV-2, as FFU/ml.

Example Embodiments

1. A compound represented by the formula

wherein R⁴ is R^(d), SO₂R^(d), C(═O)R^(d), NH C(═O)R^(d), R^(e), OR^(c),or CF₃, wherein R^(c) is H or C₁-C₁₀ hydrocarbyl, R^(d) is substitutedheterocyclic, unsubstituted heterocyclic, or unsubstituted carbocyclic,and R^(e) is substituted heteroaryl or substituted phenyl; and

-   n is 1 or 2.

2. The compound of embodiment 1, represented by the formula

wherein R⁴ is:

-   (i) C(═O)R^(d) and R^(d) is a pyrrolidonyl group,-   (ii) SO₂R^(d) and R^(d) is a piperidinyl group,-   (iii) NHC(═O)R^(d) and R^(d) is a phenyl group or a furanyl group,-   (iv) an imidazolyl group, or-   (v) a thiazolyl group.

3. The compound of embodiment 1, represented by the formula

wherein X is NH or O.

4. The compound of embodiment 1, wherein R⁴ is CF₃, OR^(c), or a phenylgroup substituted by at least One OCH₃ group.

5. A compound of claim 1 represented by the formula:

6. A compound represented by the formula

wherein L is NR², O, S, C(═O)N, CR²R³CR²R³, CR²R³NR², CR⁴═CR⁴, CR²R³O,CR²R³S, NR²CR²R³, NR²C(═O), NS(O)_(t) , OCR²R³, SCR²R³;

-   V is (CR²R³)_(u), C(═O)CR²R³, CR²R³O, CR²R³OCR²R³, CR⁴═CR⁴, C(═NR²),    or C(═O);-   Q is NR², O, S(O)_(t), or a bond;-   t=0, 1, 2; u=0-3;    wherein a dashed line indicates the presence or absence of a bond;-   R¹ is R^(a), OR², or NR²R³;-   each R^(a) is independently H, optionally substituted hydrocarbyl,    optionally substituted aryl, optionally substituted heteroaryl;-   R² and R³ are each independently R^(a), C(═O)R^(a), SO₂R^(a), or R²    and R³ form an optionally substituted carbocyclic,    heterocarbocyclic, aryl, or heteroaryl ring;-   each R⁴ is independently R², OR^(a), C(═O)R^(a), C(═O)NR²R³, NR²R³,    NR^(b)(═O)R^(a), SR^(a), SOR^(a), SO₂R^(a), SO₂NHR^(a), SO₂NR²R³,    NCOR^(a), halogen, trihalomethyl, CN, S═O, nitro, or two R⁴ groups    form an optionally substituted carbocyclic, heterocarbocyclic, aryl,    or heteroaryl ring;-   W and X are each independently N, NR^(a), NR⁵, O, S, CR²R⁴ or CR⁴;-   R⁵ is R^(a), C(═O)R^(a), SO₂R^(a), or is not present;-   Y¹, Y², Y³ and Y⁴ are each independently CR⁴ or N; and-   NR²R³ may form an optionally substituted heterocylic or heteroaryl    ring including pyrrolidine, piperidine, morpholine, and piperazine.

7. A compound of embodiment 6, the compound having a structurerepresented by Formula 1A or 1C

wherein in each of Formula 1A and Formula 1C,

-   R¹ is R^(a), OR², or NR²R³;-   each R^(a) is independently H, optionally substituted hydrocarbyl,    optionally substituted aryl, optionally substituted heteroaryl;-   R² and R³ are each independently R^(a), C(═O)R^(a), or SO₂R^(a);-   each R⁴ is independently R², OR^(a), NR²R³, SR^(a), SOR^(a),    SO₂R^(a), SO₂NHR^(a), NCOR^(a), C(═O)R^(a), CONR²R³, halogen,    trihalomethyl, CN, S═O, or nitro;-   V is CR²R³, C(═O), C(═O)CR²R³, or C(═N)R²; and-   W is O or S.

8. A compound of embodiment 6, the compound having a structurerepresented by the Formula 1B

wherein R¹ is R^(a), OR² or NR²R³;

-   each R^(a) is independently H, optionally substituted hydrocarbyl,    optionally substituted aryl, optionally substituted heteroaryl;-   R² and R³ are each independently R^(a), C(═O)R^(a), or SO₂R^(a);-   each R⁴ is independently R², OR^(a), NR²R³, SR^(a), SOR^(a),    SO₂R^(a), SO₂NHR^(a), NCOR^(a), C(═O)R^(a), CONR²R³, halogen,    trihalomethyl, CN, S═O, or nitro;-   R⁶ is H or CH₃,-   V is CR²R³, C(═O), C(═O)CR²R³, or C═NR²; and-   W is O or S.

9. A compound of embodiment 7 or 8 wherein R4 is H; and V is C═O.

-   10. A compound of any one of embodiments 7, 8, or 9 wherein R1 is    optionally substituted phenyl or optionally substituted naphthyl.-   11. A compound of any one of embodiments 6-10, wherein W is S and X    is N.-   12. A compound of any one of embodiments 6-10, wherein W is O and X    is N.-   13. A compound of any one of embodiments 6-11 represented by the    formula

wherein R¹ is a phenyl group substituted by at least one halogen, aphenyl group substituted by NR²R³, a phenyl group substituted bySO₂NR²R³, CR²R³ORd, an unsubstituted napthyl group, a napthyl groupsubstituted by O(CR²R³)_(n)R^(d), NR^(a)(CR²R³)_(n)R^(d),NR^(a)(CR²R³)_(n)NR²R³, a two membered ring structure including apyridynyl group and a phenyl group, or a two membered ring structureincluding a phenyl group and a dioxolanyl group;

-   each R^(a) is independently H or optionally substituted hydrocarbyl    (C₁-C₁₀);-   R² and R³ are each independently R^(a), COR^(a), (CH2)_(n)O, or    SO₂R^(a);-   each R⁴ is independently R^(a)-   R^(d) is phenyl or morpholino-   R⁵ is H or CH₃;-   R⁶ is H or CH₃; and-   wherein n is 1, 2, 3, or 4.

14. A compound of embodiment 13 represented by the formula

15. A compound of any one of embodiments 5-9 and 11 represented by theformula

wherein R¹ is a phenyl group substituted by at least one halogen, aphenyl group substituted by NR²R³, a phenyl group substituted bySO₂R^(d), a napthyl group optionally substituted by O(CR²R³)_(n)R^(d),or an unsubstituted napthyl group,

-   each R^(a) is independently H or optionally substituted C₁-C₁₀    hydrocarbyl;-   R², R³ and each R⁴ are independently R^(a),-   R^(d) is optionally substituted phenyl or optionally substituted    morpholino;-   R⁵ is H or CH₃;-   R⁶ is H or CH₃, and-   wherein n is 1, 2, 3, or 4.

16. A compound of embodiment 15, represented by the formula:

17. A pharmaceutical composition comprising a compound of any one ofembodiments 1 to 16.

18. A method of treating a viral infection in a subject comprisingadministering to the subject a therapeutically effective amount of apharmaceutical composition of embodiment 17 thereby treating the viralinfection in the subject.

19. A method of preventing a viral infection in a subject comprisingadministering to the subject a therapeutically effective amount of apharmaceutical composition of embodiment 17.

20. A method of embodiment 18 or embodiment 19 wherein the viralinfection is caused by a virus from one or more of the followingfamilies: Arenaviridae, Arterivirus, Astroviridae, Birnaviridae,Bromoviridae, Bunyaviridae, Caliciviridae, Closteroviridae, Comoviridae,Coronaviridae, Cystoviridae, Filoviridae, Flaviviridae, Flexiviridae,Hepadnaviridae, Hepevirus, Herpesviridae, Leviviridae, Luteoviridae,Mesoniviridae, Mononegavirales, Mosaic Viruses, Nidovirales,Nodaviridae, Orthomyxoviridae, Papillomaviridae, Paramyxoviridae,Picobirnaviridae, Picobirnavirus, Picornaviridae, Potyviridae,Reoviridae, Retroviridae, Roniviridae, Sequiviridae, Tenuivirus,Togaviridae, Tombusviridae, Totiviridae, and Tymoviridae.

21. A method of embodiment 18 or embodiment 19 wherein the viralinfection is caused by Alfuy virus, Banzi virus, bovine diarrhea virus,Chikungunya virus, Dengue virus (DNV), Hepatitis B virus (HBV),Hepatitis C virus (HCV), human cytomegalovirus (hCMV), humanimmunodeficiency virus (HIV), Ilheus virus, influenza virus (includingavian and swine isolates), rhinovirus, norovirus, adenovirus, Japaneseencephalitis virus, Kokobera virus, Kunjin virus, Kyasanur forestdisease virus, louping-ill virus, measles virus, MERS-coronavirus(MERS), metapneumovirus, any of the Mosaic Viruses, Murray Valley virus,parainfluenza virus, poliovirus, Powassan virus, respiratory syncytialvirus (RSV), Rocio virus, SARS-coronavirus (SARS), St. Louisencephalitis virus, tick-borne encephalitis virus, West Nile virus(WNV), Ebola virus, Nipah virus, Lassa virus, Tacaribe virus, Juninvirus, or yellow fever virus.

22. A pharmaceutical composition of embodiment 17, for use in therapy.

23. A pharmaceutical composition for use according to embodiment 22,wherein said pharmaceutical composition is administered as an adjuvantfor a prophylactic or therapeutic vaccine.

24. A method of modulating the innate immune response in a eukaryoticcell, comprising administering to the cell a compound of any one ofembodiments 1 to 16.

25. A method of embodiment 24, wherein the cell is in vivo.

26. A method of embodiment 25, wherein the cell is in vitro.

27. A method of treating a viral infection in a subject comprisingadministering to the subject a therapeutically effective amount of apharmaceutical composition having the structure

and wherein the viral infection is caused by Ebola virus.

As will be understood by one of ordinary skill in the art, eachembodiment disclosed herein can comprise, consist essentially of orconsist of its particular stated element, step, ingredient or component.Thus, the terms “include” or “including” should be interpreted torecite: “comprise, consist of, or consist essentially of.” As usedherein, the transition term “comprise” or “comprises” means includes,but is not limited to, and allows for the inclusion of unspecifiedelements, steps, ingredients, or components, even in major amounts. Thetransitional phrase “consisting of” excludes any element, step,ingredient or component not specified. The transition phrase “consistingessentially of” limits the scope of the embodiment to the specifiedelements, steps, ingredients or components and to those that do notmaterially affect the embodiment. As used herein, a material effectwould cause a statistically significant reduction in a disclosedcompound's or composition's ability to treat a viral infection in asubject; reduce viral protein in a subject or assay; reduce viral RNA ina subject or assay or reduce virus in a cell culture.

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as molecular weight, reaction conditions,and so forth used in the specification and claims are to be understoodas being modified in all instances by the term “about.” Accordingly,unless indicated to the contrary, the numerical parameters set forth inthe specification and attached claims are approximations that can varydepending upon the desired properties sought to be obtained by thepresent disclosure. At the very least, and not as an attempt to limitthe application of the doctrine of equivalents to the scope of theclaims, each numerical parameter should at least be construed in lightof the number of reported significant digits and by applying ordinaryrounding techniques. When further clarity is required, the term “about”has the meaning reasonably ascribed to it by a person skilled in the artwhen used in conjunction with a stated numerical value or range, i.e.denoting somewhat more or somewhat less than the stated value or range,to within a range of ±20% of the stated value; ±19% of the stated value;±18% of the stated value; ±17% of the stated value; ±16% of the statedvalue; ±15% of the stated value; ±14% of the stated value; ±13% of thestated value; ±12% of the stated value; ±11% of the stated value; ±10%of the stated value; ±9% of the stated value; ±8% of the stated value;±7% of the stated value; ±6% of the stated value; ±5% of the statedvalue; ±4% of the stated value; ±3% of the stated value; ±2% of thestated value; or ±1% of the stated value.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the disclosure are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. Any numerical value, however, inherently contains certainerrors necessarily resulting from the standard deviation found in theirrespective testing measurements.

The terms “a,” “an,” “the” and similar referents used in the context ofdescribing the disclosure (especially in the context of the followingclaims) are to be construed to cover both the singular and the plural,unless otherwise indicated herein or clearly contradicted by context.Recitation of ranges of values herein is merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”) provided herein isintended merely to better illuminate the disclosure and does not pose alimitation on the scope of the disclosure otherwise claimed. No languagein the specification should be construed as indicating any non-claimedelement essential to the practice of the disclosure.

Groupings of alternative elements or embodiments of the disclosuredisclosed herein are not to be construed as limitations. Each groupmember can be referred to and claimed individually or in any combinationwith other members of the group or other elements found herein. It isanticipated that one or more members of a group can be included in, ordeleted from, a group for reasons of convenience and/or patentability.When any such inclusion or deletion occurs, the specification is deemedto contain the group as modified thus fulfilling the written descriptionof all Markush groups used in the appended claims.

Certain embodiments of this disclosure are described herein, includingthe best mode known to the inventors for carrying out the disclosure. Ofcourse, variations on these described embodiments will become apparentto those of ordinary skill in the art upon reading the foregoingdescription. The inventor expects skilled artisans to employ suchvariations as appropriate, and the inventors intend for the disclosureto be practiced otherwise than specifically described herein.Accordingly, this disclosure includes all modifications and equivalentsof the subject matter recited in the claims appended hereto as permittedby applicable law. Moreover, any combination of the above-describedelements in all possible variations thereof is encompassed by thedisclosure unless otherwise indicated herein or otherwise clearlycontradicted by context.

Numerous references have been made to publications, patents and/orpatent applications (collectively “references”) throughout thisspecification. Each of the cited references is individually incorporatedherein by reference for their particular cited teachings.

In closing, it is to be understood that the embodiments of thedisclosure disclosed herein are illustrative of the principles of thepresent disclosure. Other modifications that may be employed are withinthe scope of the disclosure. Thus, by way of example, but not oflimitation, alternative configurations of the present disclosure may beutilized in accordance with the teachings herein. Accordingly, thepresent disclosure is not limited to that precisely as shown anddescribed.

What is claimed is:
 1. A method for treating or preventing a viral infection in a subject, said method comprising administering to said subject a pharmaceutically effective amount of a compound having the formula:

or pharmaceutically acceptable salts thereof, wherein R⁴ is R^(d), SO₂R^(d), C(═O)R^(d), NH C(═O)R^(d), R^(e), OR^(c), or CF₃, wherein R^(c) is H or C₁-C₁₀ hydrocarbyl, R^(d) is substituted heterocyclic, unsubstituted heterocyclic, or unsubstituted carbocyclic, and R^(e) is substituted heteroaryl or substituted phenyl; and n is 1 or
 2. 2. The method of claim 1, wherein the compound has the formula:

or pharmaceutically acceptable salts thereof, wherein R⁴ is: (i) C(═O)R^(d) and R^(d) is a pyrrolidonyl group, (ii) SO₂R^(d) and R^(d) is a piperidinyl group, (iii) NHC(═O)R^(d) and R^(d) is a phenyl group or a furanyl group, (iv) an imidazolyl group, or (v) a thiazolyl group.
 3. The method of claim 1, wherein the compound has the formula:

or pharmaceutically acceptable salts thereof wherein X is NH or
 0. 4. The method of claim 1, wherein R⁴ is CF₃, OR^(c), or a phenyl group substituted by at least one OCH₃ group.
 5. The method of claim 1, wherein the compound has the formula:


6. A method for treating or preventing a viral infection in a subject, said method comprising administering to said subject a pharmaceutically effective amount of a compound having the formula:

or pharmaceutically acceptable salts thereof, wherein L is NR², O, S, C(═O)N, CR²R³CR²R³, CR²R³NR², CR⁴═CR⁴, CR²R³O, CR²R³S, NR²CR²R³, NR²C(═O), NS(O)_(t) , OCR²R³, SCR²R³; V is (CR²R³)_(u), C(═O)CR²R³, CR²R³O, CR²R³OCR²R³, CR⁴═CR⁴, C(=NR²), or C(═O); Q is NR², O, S(O)_(t), or a bond; t=0, 1, 2; u=0-3; wherein a dashed line indicates the presence or absence of a bond; R¹ is R^(a), OR², or NR²R³; each R^(a) is independently H, optionally substituted hydrocarbyl, optionally substituted aryl, optionally substituted heteroaryl; R² and R³ are each independently R^(a), C(═O)R^(a), SO₂R^(a), or R² and R³ form an optionally substituted carbocyclic, heterocarbocyclic, aryl, or heteroaryl ring; each R⁴ is independently R², OR^(a), C(═O)R^(a), C(═O)NR²R³, NR²R³, NR^(b)(═O)R^(a), SR^(a), SOR^(a), SO₂R^(a), SO₂NHR^(a), SO₂NR²R³, NCOR^(a), halogen, trihalomethyl, CN, S═O, nitro, or two R⁴ groups form an optionally substituted carbocyclic, heterocarbocyclic, aryl, or heteroaryl ring; W and X are each independently N, NR^(a), NR^(S), O, S, CR²R⁴ or CR⁴; R⁵ is R^(a), C(═O)R^(a), SO₂R^(a), or is not present; and Y¹, Y², Y³ and Y⁴ are each independently CR⁴ or N.
 7. The method of claim 6 wherein the compound has the formula:

or pharmaceutically acceptable salts thereof, wherein in each of Formula 1A and Formula 1C, R¹ is R^(a), OR², or NR²R³; each R^(a) is independently H, optionally substituted hydrocarbyl, optionally substituted aryl, optionally substituted heteroaryl; R² and R³ are each independently R^(a), C(═O)R^(a), or SO₂R^(a); each R⁴ is independently R², OR^(a), NR²R³, SR^(a), SOR^(a), SO₂R^(a), SO₂NHR^(a), NCOR^(a), C(═O)R^(a), CONR²R³, halogen, trihalomethyl, CN, S═O, or nitro; V is CR²R³, C(═O), C(═O)CR²R³, or C(═N)R²; and W is O or S.
 8. The method of claim 7 wherein R⁴ is H; and V is C═O.
 9. The method of claim 7 wherein R¹ is optionally substituted phenyl or optionally substituted naphthyl.
 10. The method of claim 6, wherein W is S and X is N.
 11. The method of claim 6, wherein W is O and X is N.
 12. The method of claim 1 wherein the compound has the formula:

or pharmaceutically acceptable salt thereof wherein R⁴ is optionally substituted heteroaryl.
 13. The method of claim 1 wherein the compound has the formula:

or pharmaceutically acceptable salt thereof.
 14. The method of claim 1 wherein the viral infection is caused by a virus from one or more of the following families: Arenaviridae, Arterivirus, Astroviridae, Birnaviridae, Bromoviridae, Bunyaviridae, Caliciviridae, Closteroviridae, Comoviridae, Coronaviridae, Cystoviridae, Filoviridae, Flaviviridae, Flexiviridae, Hepadnaviridae, Hepevirus, Herpesviridae, Leviviridae, Luteoviridae, Mesoniviridae, Mononegavirales, Mosaic Viruses, Nidovirales, Nodaviridae, Orthomyxoviridae, Papillomaviridae, Paramyxoviridae, Picobirnaviridae, Picobirnavirus, Picornaviridae, Potyviridae, Reoviridae, Retroviridae, Roniviridae, Sequiviridae, Tenuivirus, Togaviridae, Tombusviridae, Totiviridae, and Tymoviridae.
 15. The method of claim 6 wherein the viral infection is caused by a virus from one or more of the following families: Arenaviridae, Arterivirus, Astroviridae, Birnaviridae, Bromoviridae, Bunyaviridae, Caliciviridae, Closteroviridae, Comoviridae, Coronaviridae, Cystoviridae, Filoviridae, Flaviviridae, Flexiviridae, Hepadnaviridae, Hepevirus, Herpesviridae, Leviviridae, Luteoviridae, Mesoniviridae, Mononegavirales, Mosaic Viruses, Nidovirales, Nodaviridae, Orthomyxoviridae, Papillomaviridae, Paramyxoviridae, Picobirnaviridae, Picobirnavirus, Picornaviridae, Potyviridae, Reoviridae, Retroviridae, Roniviridae, Sequiviridae, Tenuivirus, Togaviridae, Tombusviridae, Totiviridae, and Tymoviridae.
 16. The method of claim 1, wherein the viral infection is caused by Alfuy virus, Banzi virus, bovine diarrhea virus, Chikungunya virus, Dengue virus (DNV), Hepatitis B virus (HBV), Hepatitis C virus (HCV), human cytomegalovirus (hCMV), human immunodeficiency virus (HIV), Ilheus virus, influenza virus (including avian and swine isolates), rhinovirus, norovirus, adenovirus, Japanese encephalitis virus, Kokobera virus, Kunjin virus, Kyasanur forest disease virus, louping-ill virus, measles virus, MERS-coronavirus (MERS), metapneumovirus, any of the Mosaic Viruses, Murray Valley virus, parainfluenza virus, poliovirus, Powassan virus, respiratory syncytial virus (RSV), Rocio virus, SARS-coronavirus (SARS), St. Louis encephalitis virus, tick-borne encephalitis virus, West Nile virus (WNV), Ebola virus, Nipah virus, Lassa virus, Tacaribe virus, Junin virus, or yellow fever virus.
 17. The method of claim 6, wherein the viral infection is caused by Alfuy virus, Banzi virus, bovine diarrhea virus, Chikungunya virus, Dengue virus (DNV), Hepatitis B virus (HBV), Hepatitis C virus (HCV), human cytomegalovirus (hCMV), human immunodeficiency virus (HIV), Ilheus virus, influenza virus (including avian and swine isolates), rhinovirus, norovirus, adenovirus, Japanese encephalitis virus, Kokobera virus, Kunjin virus, Kyasanur forest disease virus, louping-ill virus, measles virus, MERS-coronavirus (MERS), metapneumovirus, any of the Mosaic Viruses, Murray Valley virus, parainfluenza virus, poliovirus, Powassan virus, respiratory syncytial virus (RSV), Rocio virus, SARS-coronavirus (SARS), St. Louis encephalitis virus, tick-borne encephalitis virus, West Nile virus (WNV), Ebola virus, Nipah virus, Lassa virus, Tacaribe virus, Junin virus, or yellow fever virus.
 18. The method of claim 1 wherein the viral infection is an RNA virus.
 19. The method of claim 6 wherein the viral infection is an RNA virus.
 20. The method of claim 1 wherein the viral infection is a DNA virus.
 21. The method of claim 6 wherein the viral infection is a DNA virus.
 22. The method of claim 1 wherein the viral infection is caused by Coronaviridae virus.
 23. The method of claim 6 wherein the viral infection is caused by Coronaviridae virus.
 24. The method of claim 1 wherein the viral infection is caused by MERS-coronavirus (MERS) or by SARS-coronavirus (SARS).
 25. The method of claim 6 wherein the viral infection is caused by MERS-coronavirus (MERS) or by SARS-coronavirus (SARS). 